Human Anti-Alpha-Synuclein Antibodies

ABSTRACT

Provided are novel human α-synuclein-specific antibodies as well as fragments, derivatives and variants thereof as well as methods related thereto. Assays, kits, and solid supports related to antibodies specific for α-synuclein are also disclosed. The antibody, immunoglobulin chain(s), as well as binding fragments, derivatives and variants thereof can be used in pharmaceutical and diagnostic compositions for α-synuclein targeted immunotherapy and diagnosis, respectively.

FIELD OF THE INVENTION

The present invention generally relates to novel α-synuclein-specificbinding molecules, particularly human antibodies as well as fragments,derivatives and variants thereof that recognize α-synuclein andaggregated forms of α-synuclein, respectively. In addition, the presentinvention relates to pharmaceutical and diagnostic compositionscomprising such binding molecules, antibodies and mimics thereofvaluable both as a diagnostic tool to identify toxic species ofα-synuclein in plasma and CSF and also in passive vaccination strategiesfor treating disorders related to aggregates of α-synuclein such asParkinson's disease (PD), dementia with Lewy bodies (DLB) and Lewy bodyvariant of Alzheimer's disease (AD) and other synucleinopathic diseases.

BACKGROUND OF THE INVENTION

Protein misfolding and aggregation are pathological aspects of numerousneurodegenerative diseases. Aggregates of α-synuclein are majorcomponents of the Lewy bodies and Lewy neurites associated withParkinson's disease (PD). A natively unfolded protein, α-synuclein canadopt different aggregated morphologies, including oligomers,protofibrils and fibrils. The small oligomeric aggregates have beenshown to be particularly toxic.

Naturally occurring autoantibodies against α-synuclein have beendetected in healthy persons and altered levels in patients wereassociated with particular neurodegenerative disorders; see for reviewNeff et al., Autoimmun. Rev. 7 (2008), 501-507. Thus, naturallyoccurring antibodies in patients suffering from Parkinson's disease,either spontaneously or upon vaccination, in particular in healthypatients may serve a protective role with respect to α-synucleinaggregation; see, e.g., Woulfe et al., Neurology 58 (2002), 1435-1436and Papachroni et al., J. Neurochem. 101 (2007), 749-756. Hitherto, thetherapeutic significance of autoantibodies had been difficult to assess.This is mostly due to the lack of straight-forward experimentalapproaches for their isolation and subsequent characterization in vitro.

Recently, oligomeric species of α-synuclein have been reportedextracellularly in plasma and CSF (El-Agnaf et al., FASEB J. 20 (2006),419-425) and immunization studies in mouse models of PD show thatextracellular mouse monoclonal antibodies against α-synuclein can reduceaccumulation of intracellular α-synuclein aggregates (Masliah et al.,Neuron, 46 (2005), 857-868) supporting the idea that antibodies thatneutralize the neurotoxic aggregates without interfering with beneficialfunctions of monomeric α-synuclein may be useful therapeutics. However,the therapeutic utility of murine based antibodies in human is hamperedby the human anti-mouse antibody (HAMA) response in view of theirnon-human origin.

Emadi et al. in J. Mol. Biol. 368 (2007), 1132-1144, describe theisolation of single chain antibody fragments (scFvs) from a phagedisplayed antibody library based on human sequences against α-synuclein,which bind only to an oligomeric form of α-synuclein and inhibit bothaggregation and toxicity of α-synuclein in vitro. However, although thegeneration of scFvs from phage display is rather simple, this techniquehas severe drawbacks since the antibodies so produced bear the risk ofundesired crossreactivity against self-antigens and lack thecharacteristics of evolutionary optimized natural human antibodiesproduced by the human immune system. Furthermore, such antibodies maynot be specific enough because of cross-reactivity with other proteinsand/or with the target protein in context with normal physiologicalenvironment and function. In case of Parkinson's disease, for example,antibodies that also cross-react with physiological derivatives ofα-synuclein bear the potential to cause side effects related to thenormal functions of the physiologic target structures. In this respect,an undesired autoimmune disease would downrightly be induced—a hardlycalculable risk also in the conceptual design of active immunizationexperiments employing protein structures that, in variant form, alsooccur physiologically.

More recently, Seitz et al. (81. Kongress der Deutschen Gesellschaft fürNeurologie mit Fortbildungsakademie Hamburg 10.-13.09.2008), reported onthe isolation of anti-α-synuclein polyclonal autoantibody from differentimmunoglobulin solutions and samples of single blood donors throughaffinity chromatography. However, besides the fact that this approachprovides mere limited amounts of the desired antibody, polyclonalantibodies are of only limited use for therapeutic application, forexample because of their heterogeneity and the risk of beingcontaminated with other α-synuclein associated molecules which haveundesired side effects. Likewise, the diagnostic value of polyclonalantibodies is reduced since the variability of the composition of theantibodies will influence the overall specificity and reactivity. Thisis all the more true for antibodies against proteins subject ofaggregation and deposition due to misfolding.

Thus, there is a need to overcome the above-described limitations and toprovide a therapeutic and diagnostic human antibody against α-synuclein.

SUMMARY OF THE INVENTION

The present invention makes use of the α-synuclein-specific immuneresponse of aged healthy control subjects and patients with neurologicaldisease for the isolation of natural α-synuclein specific humanmonoclonal antibodies. In particular, experiments performed inaccordance with the present invention were successful in the isolationof monoclonal antibodies specific for α-synuclein from a pool of elderlysubjects with no signs of Parkinsonism.

The present invention is thus directed to human antibodies,antigen-binding fragments and similar antigen-binding molecules whichare capable of specifically recognizing α-synuclein. By “specificallyrecognizing α-synuclein”, “antibody specific to/for α-synuclein” and“anti-α-synuclein antibody” is meant specifically, generally, andcollectively, antibodies to the native form of α-synuclein, or misfoldedor oligomeric or aggregated or posttranslationally modified α-synuclein.Provided herein are human antibodies selective for native monomer,full-length, truncated and aggregated forms.

In a particularly preferred embodiment of the present invention, thehuman antibody or antigen-binding fragment thereof demonstrates theimmunological binding characteristics of an antibody characterized bythe variable regions V_(H) and/or V_(L) as set forth in FIG. 1.

The antigen-binding fragment of the antibody can be a single chain Fvfragment, an F(ab′) fragment, an F(ab) fragment, and an F(a′)₂ fragment,or any other antigen-binding fragment. In a specific embodiment, infra,the antibody or fragment thereof is a human IgG isotype antibody.Alternatively, the antibody is a chimeric human-murine or murinizedantibody, the latter being particularly useful for diagnostic methodsand studies in animals.

Furthermore, the present invention relates to compositions comprisingthe antibody of the present invention or active fragments thereof, oragonists and cognate molecules, or alternately, antagonists of the sameand to immunotherapeutic and immunodiagnostic methods using suchcompositions in the prevention, diagnosis or treatment of asynucleinopathic disease, wherein an effective amount of the compositionis administered to a patient in need thereof.

Naturally, the present invention extends to the immortalized human Bmemory lymphocyte and B cell, respectively, that produces the antibodyhaving the distinct and unique characteristics as defined below.

The present invention also relates to polynucleotides encoding at leasta variable region of an immunoglobulin chain of the antibody of theinvention. Preferably, said variable region comprises at least onecomplementarity determining region (CDR) of the V_(H) and/or V_(L) ofthe variable region as set forth in FIG. 1.

Accordingly, the present invention also encompasses vectors comprisingsaid polynucleotides and host cells transformed therewith as well astheir use for the production of an antibody and equivalent bindingmolecules which are specific for α-synuclein. Means and methods for therecombinant production of antibodies and mimics thereof as well asmethods of screening for competing binding molecules, which may or maynot be antibodies, are known in the art. However, as described herein,in particular with respect to therapeutic applications in human theantibody of the present invention is a human antibody in the sense thatapplication of said antibody is substantially free of a HAMA responseotherwise observed for chimeric and even humanized antibodies.

Furthermore, disclosed herein are compositions and methods that can beused to identify α-synuclein in samples. The disclosed anti-α-synucleinantibodies can be used to screen human blood, CSF, and urine for thepresence of α-synuclein in samples, for example, by using ELISA-based orsurface adapted assay. The methods and compositions disclosed herein canaid in synucleinopathic disease such as Parkinson's disease diagnosisand can be used to monitor disease progression and therapeutic efficacy.

As demonstrated in Example 4, the anti-α-synuclein antibody of thepresent invention is capable of improving motor performance and elevatedplus maze behavior in a transgenic mouse model of Parkinson's disease.These results confirm the expected therapeutic value of thehuman-derived anti-α-synuclein antibodies of the present invention.

Hence, it is a particular object of the present invention to providemethods for treating or preventing a synucleinopathic disease such asParkinson's disease (PD), Parkinson's Disease Dementia (PDD), dementiawith Lewy bodies (DLB), the Lewy body variant of Alzheimer's disease(LBVAD), multiple systems atrophy (MSA), pure autonomic failure (PAF),neurodegeneration with brain iron accumulation type-1 (NBIA-I),Alzheimer's disease, Pick disease, juvenile-onset generalizedneuroaxonal dystrophy (Hallervorden-Spatz disease), amyotrophic lateralsclerosis, traumatic brain injury and Down syndrome. The methodscomprise administering an effective concentration of a human antibody orantibody derivative to the subject where the antibody targetsα-synuclein.

Further embodiments of the present invention will be apparent from thedescription and Examples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Amino acid and nucleotide sequences of the variable region, i.e.heavy chain and kappa/lambda light chain of human antibodies NI-202.3G12(A), NI-202.12F4 (B) and NI-202.3D8 (C). For human antibody NI-202.3D8two variable light chain sequences VKal (C) and VKcl (D) have beencloned, each of which may be paired with the variable heavy chainsequence VHE1 (C). Framework (FR) and complementarity determiningregions (CDRs) are indicated with the CDRs being underlined. The heavychain joining region (JH) and light chain joining region (JK) areindicated as well. Due to the cloning strategy the amino acid sequenceat the N-terminus of the heavy chain and light chain may potentiallycontain primer-induced alterations in FR1, which however do notsubstantially affect the biological activity of the antibody. In orderto provide a consensus human antibody, the nucleotide and amino acidsequences of the original clone were aligned with and tuned inaccordance with the pertinent human germ line variable region sequencesin the database; see, e.g., Vbase (http://vbase.mrc-cpe.cam.ac.uk/)hosted by the MRC Centre for Protein Engineering (Cambridge, UK). Thoseamino acids, which are considered to potentially deviate from theconsensus germ line sequence and thus could be due to the PCR primer,are indicated in bold.

FIG. 2: Recombinant human α-synuclein antibodies are directed againstdistinct epitopes. Recombinant full-length and truncated α-synuclein wascoated onto ELISA plates at equal coating concentration (20 μg/ml). (A)Recombinant human α-synuclein antibody NI-202.3G12 binds to full lengthα-synuclein but not to α-synuclein truncations in a direct ELISA,pointing to a structural recognition epitope of NI-202.3G12. (B)Recombinant NI-202.12F4 binds to full length α-synuclein and toα-synuclein truncations containing amino acids (aa) 1-60 in a directELISA, pointing to an epitope of NI-202.12F4 within the N-terminalamphipathic repeat region of alpha synuclein. (C) LB509 antibody bindsto C-terminal α-synuclein fragments confirming the previously determinedepitope (aa 115-122). Values are means±SEM (n=2−3).

FIG. 3: Recombinant human α-synuclein antibodies bind α-synuclein butnot β- and γ-synuclein in a direct ELISA. Recombinant α-, β- andγ-synuclein coated onto ELISA plates at equal coating concentration (2μg/ml) were incubated with recombinant human α-synuclein antibodies orwith a pan synuclein antibody. (A) The latter detects all threesynuclein proteins whereas recombinant human α-synuclein antibodies (B)NI-202.3G12 and (C) NI-202.12F4 (C) selectively bind to α-synuclein.Values are means±SEM (n=2−3).

FIG. 4: Recombinant human α-synuclein antibody NI-202.12F4 bindsα-synuclein but not β- and γ-synuclein on Western blot analysis.Recombinant α-, β- and γ-synuclein (each 750 ng) were subjected toSDS-PAGE and subsequent to Western Blot analysis. (A) Coomassie stainingreveals equal protein concentration on SDS-PAGE. (B) NI-202.12F4strongly interacts with α-synuclein but not with beta or gammasynuclein. (C) No signal was detected without primary antibody.

FIG. 5: (A) NI-202.12F4 immunoblot analysis of brain extracts fromnon-transgenic and human alpha-synuclein transgenic mice showspreferential binding to human α-synuclein. Brain extracts from wild typeand human α-synuclein transgenic mice were analyzed by immunoblottingwith human specific α-synuclein antibody LB509, human and mouseα-synuclein reactive antibody clone 42 and NI-202.12F4. While clone 42detects prominent bands corresponding to mouse and human α-synuclein,LB509 and NI-202.12F4 show a strong preference for human α-synuclein.(B) NI-202.12F4 immunoblot analysis of brain extracts shows preferentialbinding to human α-synuclein aggregates. Cortical brain extracts from ahealthy control subject and a dementia with Lewy bodies (DLB) patient aswell as brain extracts from wild type mice and human A30P α-synucleintransgenic mice were analyzed by Western blotting. NI-202.12F4 detectsoligomeric and fibrillar forms of α-synuclein aggregates in DLB and A30Pα-synuclein transgenic brain extract with high sensitivity. Minimalbinding is observed to monomeric forms of α-synuclein in human or wildtype mouse tissues and moderate binding in A30P α-synuclein transgenicbrain extracts highly overexpressing α-synuclein. In contrast, clone 42antibody detects monomeric forms and α-synuclein fragments with a highsensitivity and poorly binds to aggregated α-synuclein species.

FIG. 6: Recombinant NI-202.12F4 shows high affinity binding to wild typeand disease causing mutants of human α-synuclein. Recombinant wild-type(●), A53T (▪), A30P (*) and E64K (⋄) human α-synuclein were coated onELISA plates (2 μg/ml) and probed with various concentrations ofNI202.12F4. Half maximal effective concentrations (EC50) were 321pM forwild-type α-synuclein, 293pM for A53T, 228 pM for A30P and 483 pM forE64K mutant human α-synuclein.

FIG. 7: Immunohistochemical binding analysis of NI-202.12F4. NI-202.12F4shows prominent staining of α-synuclein pathology including Lewy bodyand Lewy neurite like inclusion as well as small somatodendritic andsynaptic α-synuclein accumulations in free-floating sections fromtransgenic mice expressing human A53T (A) or A30P α-synuclein (B) aswell as in human brain tissues of Parkinson's disease (C) and dementiawith Lewy bodies (D). Antibody Syn211 detects physiological synapticα-synuclein with a high sensitivity in human A30P α-synuclein transgenicmice (E) while NI-202.12F4 binds preferentially to pathologicalα-synuclein aggregates (B). Binding of NI-202.12F4 is virtually absentfrom brain sections of wild type mice (F) comparable to secondaryantibody only control staining (G) while clone 42 antibody showsprominent synaptic staining of mouse α-synuclein protein (H).

FIG. 8: Recombinant NI-202.12F4 shows preferential binding to highdensity coated α-synuclein. Recombinant full-length or truncatedα-synuclein were coated on ELISA plates at the indicated concentrationsand probed with various concentrations of NI-202.12F4 or Syn211antibodies by direct ELISA (◯20 μg/ml; ▴2 μg/ml; ▾1 μg/ml; ♦250 ng/ml;

100 ng/ml coating concentration of recombinant full length or 1-60α-synuclein). The half maximal effective concentration (EC50) indicatingthe potency of the antibodies was determined. (A) High affinity bindingof recombinant NI-202.12F4 to α-synuclein requires high coatingdensities of α-synuclein protein. While a 111 pM EC50 is observed forα-synuclein coated at 20 μg/ml concentration EC50 values increasesharply with decreasing coating concentrations demonstrating a dramaticloss in affinity at lower coating concentrations of α-synuclein. Thesefeatures are pointing to a conformational epitope of NI-202.12F4 that ispreferentially formed at high coating concentrations of α-synuclein. (B)Binding of Syn211 is not affected by the coating concentration. Nodecrease in affinity is observed of the commercially available Syn211antibody at lower coating densities with EC50s ranging from 335 pm for20 μg/ml to 99 pM for 100 ng/ml coating density of α-synuclein,suggesting binding to a linear non-conformational epitope. (C) Highaffinity binding of recombinant NI-202.12F4 to N-terminal α-synucleinfragment comprising amino acids 1-60 requires high coating densities.NI-202.12F4 shows equivalent and coating concentration dependent bindingto full-length as well as truncatedα-synuclein pointing to aconformational epitope of NI-202.12F4 that is contained within aminoacids 1-60 of the α-synuclein protein. (D) Biotinylated peptidescomprising overlapping 20 amino acid fragments covering the N-terminal60 amino acids of α-synuclein were coated on avidin plates and probedwith NI-202.12F4 or a pan-synuclein control antibody that detects anepitope within aa 21-40. Accordingly, the control antibody stronglybinds peptide 21-40 and to lesser extent peptides 11-30 and 31-50. Incontrast, no binding is observed for NI-202.12F4 to any of the peptidestested, suggesting that none of the N-terminal fragments is sufficientas NI-202.12F4 epitope and a larger fragment may be required for optimalbinding and formation of the structural NI-202.12F4 epitope.

FIG. 9: Chronic treatment with NI-202.12F4 improves motor performance inα-synuclein A53T transgenic mice. 10.5 month old α-synuclein A53Ttransgenic mice were treated weekly with NI-202.12F4 or PBS (5 mg/kg;intraperitoneal application). Motor performance was assessed after twomonths of treatment in the Pole-Test. (A) NI-202.12F4 treated animalsrequired significantly less time to turn downwards (t-turn; 1.7±0.3 vs.4.6±0.6 sec, p=0.0002, two-tailed Student's t-test). (B) NI-202.12F4treated animals also used significantly less time (t-total) to descendto the home cage (7.3±0.9 vs. 10.4±0.7 sec, p=0.012, two-tailedStudent's t-test).

FIG. 10: Chronic treatment of α-synuclein A53T transgenic mice withNI-202.12F4 leads to recovery of elevated plus maze behavior. 10.5 monthold α-synuclein A53T transgenic mice were treated weekly i.p. with 5mg/kg NI-202.12F4 or PBS. After two month of treatment, animals weretested for elevated plus maze behavior. NI-202.12F4 treated animalsspend significantly less time and covered significantly lower distancein the open arms compared to vehicle treated control animals indicatinga recovery of normal behavior.

FIG. 11: Chronic treatment of α-synuclein A53T transgenic mice withNI-202.12F4 results in elevated plasma levels of human A53T α-synuclein.Plasma samples were prepared from 12.5 month old α-synuclein A53Ttransgenic mice that had been treated weekly i.p. for 2 months with 5mg/kg NI-202.12F4 or PBS. Blood was taken 24 hrs after last application.(A) NI-202.12F4 levels in plasma were determined using a directα-synuclein ELISA. (B) Human A53T α-synuclein levels in plasma weredetermined using a human-specific α-synuclein sandwich ELISA. Animalstreated with NI-202.12F4 have significantly elevated levels of humanα-synuclein in plasma compared to control animals (24.9±4.1 vs. 1.9±1.2ng/ml, p=0.0002).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Synucleinopathic diseases or synucleinopathies are a diverse group ofneurodegenerative disorders that share a common pathologic lesioncomposed of aggregates of insoluble α-synuclein protein in selectivelyvulnerable populations of neurons and glia. These disorders includeParkinson's disease (PD), Parkinson's Disease Dementia (PDD), dementiawith Lewy bodies (DLB), juvenile-onset generalized neuroaxonal dystrophy(Hallervorden-Spatz disease), pure autonomic failure (PAF), multiplesystem atrophy (MSA) and neurodegeneration with brain iron accumulationtype-1 (NBIA-I). Clinically, they are characterized by a chronic andprogressive decline in motor, cognitive, behavioral, and autonomicfunctions, depending on the distribution of the lesions.

Parkinson's disease is an age-dependent neurodegenerative disease withunknown etiology. It is believed that sporadic Parkinson's diseaseresults from a combination of genetic vulnerability and environmentalinsults. It is further believed that Parkinson's disease (PD) whiletriggered by disparate mechanisms follows a shared pathophysiologicpathway. One shared node is the involvement of α-synuclein. Linkage ofthis protein with Parkinson's disease pathogenesis has been establishedby the identification of both point mutations and triplication of thegene in familial cases, the localization of α-synuclein to Lewy bodies,one of the hallmark pathological features of Parkinson's disease, andthe correlation of α-synuclein expression and disease pathology inneurotoxic models of Parkinson's disease. Further evidence indicatesthat particular forms of α-synuclein (e.g., misfolded and α-synucleinbonded dopamine) are involved in sporadic disease.

Synucleins are small, soluble proteins expressed primarily in neuraltissue and in certain tumors. The family includes three known proteins:α-synuclein, β-synuclein, and γ-synuclein. All synucleins have in commona highly conserved α-helical lipid-binding motif with similarity to theclass-A2 lipid-binding domains of the exchangeable apolipoproteins.Synuclein family members are not found outside vertebrates, althoughthey have some conserved structural similarity with plant‘late-embryo-abundant’ proteins. The α- and β-synuclein proteins arefound primarily in brain tissue, where they are seen mainly inpresynaptic terminals. The γ-synuclein protein is found primarily in theperipheral nervous system and retina, but its expression in breasttumors is a marker for tumor progression. Normal cellular functions havenot been determined for any of the synuclein proteins, although somedata suggest a role in the regulation of membrane stability and/orturnover. Mutations in α-synuclein are associated with rare familialcases of early-onset Parkinson's disease, and the protein accumulatesabnormally in Parkinson's disease, Alzheimer's disease, and severalother neurodegenerative illnesses. For review see, e.g., George, GenomeBiol. 3 (2002), reviews3002.1—reviews3002.6 published online Dec. 20,2001, in which Table 1 catalogs the unique members of the synucleinfamily that are currently listed in GenBank, the disclosure content ofwhich is incorporated herein by reference.

α-synuclein was originally identified in human brains as the precursorprotein of the non-β-amyloid component of (NAC) of Alzheimer's disease(AD) plaques; see, e.g., Ueda et al, Proc. Natl. Acad. Sci. U.S.A. 90(1993), 1282-1286. α-synuclein, also termed the precursor of the non-Aβcomponent of AD amyloid (NACP), is a protein of 140 amino acids.α-synuclein exists in its native form as a random coil; however, changesin pH, molecular crowding, heavy metal content, and dopamine levels allaffect protein conformation. Changes in conformation to oligomeric,proto-fibrillar, fibrillar, and aggregate moieties are thought toregulate protein toxicity. Increasing evidence indicates thatdopamine-adducted α-synuclein has a faster time course to fibrilformation compared to non-adducted protein. Furthermore, dopamine in thebackground of α-synuclein overexpression is toxic.

In this specification, the terms “α-synuclein”, “alphα-synuclein”,“α-synuclein” and “aSyn” are used interchangeable to specifically referto the native monomer form of α-synuclein. The term “α-synuclein” isalso used to generally identify other conformers of α-synuclein, forexample, α-synuclein bonded to dopamine-quinone (DAQ) and oligomers oraggregates of α-synuclein. The term “α-synuclein” is also used to refercollectively to all types and forms of α-synuclein. The protein sequencefor human α-synuclein isMDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA (SEQ ID NO: 1). The amino acid sequenceof α-synuclein can be retrieved from the literature and pertinentdatabases; see, e.g., Ueda et al., ibid.; GenBank swissprot: locusSYUA_HUMAN, accession number P37840.

The non-Aβ component of AD amyloid (NAC) is derived from α-synuclein.NAC, a highly hydrophobic domain within α-synuclein, is a peptideconsisting of at least 28 amino acids residues (residues 60-87) andoptionally 35 amino acid residues (residues 61-95). NAC displays atendency to form a beta-sheet structure (Iwai, et al., Biochemistry, 34(1995) 10139-10145). The amino acid sequences of NAC are described inJensen et al., Biochem. J. 310 (1995), 91-94; GenBank accession numberS56746 and Ueda et al., PNAS USA 90 (1993), 1282-11286.

Disaggregated α-synuclein or fragments thereof, including NAC, meansmonomeric peptide units. Disaggregated α-synuclein or fragments thereofare generally soluble, and are capable of self-aggregating to formsoluble oligomers. Oligomers of α-synuclein and fragments thereof areusually soluble and exist predominantly as α-helices. Monomericα-synuclein may be prepared in vitro by dissolving lyophilized peptidein neat DMSO with sonication. The resulting solution is centrifuged toremove any insoluble particulates. Aggregated α-synuclein or fragmentsthereof, including NAC, means oligomers of α-synuclein or fragmentsthereof which have associate into insoluble β-sheet assemblies.Aggregated α-synuclein or fragments thereof, including NAC, means alsomeans fibrillar polymers. Fibrils are usually insoluble. Some antibodiesbind either soluble α-synuclein or fragments thereof or aggregatedα-synuclein or fragments thereof. Some antibodies bind to oligomers ofα-synuclein more strongly than to monomeric forms or fibrillar forms.Some antibodies bind both soluble and aggregated α-synuclein orfragments thereof, and optionally oligomeric forms as well.

The human anti-α-synuclein antibodies disclosed herein specifically bindα-synuclein and epitopes thereof and to various conformations ofα-synuclein and epitopes thereof. For example, disclosed herein areantibodies that specifically bind α-synuclein, α-synuclein in its nativemonomer form, full-length and truncated α-synuclein and α-synucleinaggregates. As used herein, reference to an antibody that “specificallybinds”, “selectively binds”, or “preferentially binds” α-synucleinrefers to an antibody that does not bind other unrelated proteins. Inone example, an α-synuclein antibody disclosed herein can bindα-synuclein or an epitope thereof and show no binding above about 1.5times background for other proteins. An antibody that “specificallybinds” or “selectively binds” α-synuclein conformer refers to anantibody that does not bind all conformations of α-synuclein, i.e., doesnot bind at least one other α-synuclein conformer. For example,disclosed herein are antibodies that can distinguish among monomeric andaggregated forms of α-synuclein, human and mouse α-synuclein;full-length α-synuclein and truncated forms as well as human α-synucleinversus β- and γ-synuclein. Since the human anti-α-synuclein antibodiesof the present invention have been isolated from a pool of elderlysubjects with no signs of Parkinsonism and exhibiting anα-synuclein-specific immune response the anti-α-synuclein antibodies ofthe present invention may also be called “human auto-antibodies” inorder to emphasize that those antibodies were indeed expressed by thesubjects and have not been isolated from, for example a humanimmunoglobulin expressing phage library, which hitherto represented onecommon method for trying to provide human-like antibodies.

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “an antibody,” is understood to representone or more antibodies. As such, the terms “a” (or “an”), “one or more,”and “at least one” can be used interchangeably herein.

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and refers to amolecule composed of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids, and does not refer to aspecific length of the product. Thus, peptides, dipeptides, tripeptides,oligopeptides, “protein,” “amino acid chain,” or any other term used torefer to a chain or chains of two or more amino acids, are includedwithin the definition of “polypeptide,” and the term “polypeptide” maybe used instead of, or interchangeably with any of these terms.

The term “polypeptide” is also intended to refer to the products ofpost-expression modifications of the polypeptide, including withoutlimitation glycosylation, acetylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, or modification by non-naturally occurring amino acids. Apolypeptide may be derived from a natural biological source or producedby recombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. It may be generated in any manner,including by chemical synthesis.

A polypeptide of the invention may be of a size of about 3 or more, 5 ormore, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 ormore, 200 or more, 500 or more, 1,000 or more, or 2,000 or more aminoacids. Polypeptides may have a defined three-dimensional structure,although they do not necessarily have such structure. Polypeptides witha defined three-dimensional structure are referred to as folded, andpolypeptides which do not possess a defined three-dimensional structure,but rather can adopt a large number of different conformations, and arereferred to as unfolded. As used herein, the term glycoprotein refers toa protein coupled to at least one carbohydrate moiety that is attachedto the protein via an oxygen-containing or a nitrogen-containing sidechain of an amino acid residue, e.g., a serine residue or an asparagineresidue.

By an “isolated” polypeptide or a fragment, variant, or derivativethereof is intended a polypeptide that is not in its natural milieu. Noparticular level of purification is required. For example, an isolatedpolypeptide can be removed from its native or natural environment.Recombinantly produced polypeptides and proteins expressed in host cellsare considered isolated for purposed of the invention, as are native orrecombinant polypeptides which have been separated, fractionated, orpartially or substantially purified by any suitable technique.

Also included as polypeptides of the present invention are fragments,derivatives, analogs, or variants of the foregoing polypeptides, and anycombination thereof. The terms “fragment,” “variant,” “derivative” and“analog” when referring to antibodies or antibody polypeptides of thepresent invention include any polypeptides which retain at least some ofthe antigen-binding properties of the corresponding native bindingmolecule, antibody, or polypeptide. Fragments of polypeptides of thepresent invention include proteolytic fragments, as well as deletionfragments, in addition to specific antibody fragments discussedelsewhere herein. Variants of antibodies and antibody polypeptides ofthe present invention include fragments as described above, and alsopolypeptides with altered amino acid sequences due to amino acidsubstitutions, deletions, or insertions. Variants may occur naturally orbe non-naturally occurring. Non-naturally occurring variants may beproduced using art-known mutagenesis techniques. Variant polypeptidesmay comprise conservative or non-conservative amino acid substitutions,deletions or additions. Derivatives of α-synuclein specific bindingmolecules, e.g., antibodies and antibody polypeptides of the presentinvention, are polypeptides which have been altered so as to exhibitadditional features not found on the native polypeptide. Examplesinclude fusion proteins. Variant polypeptides may also be referred toherein as “polypeptide analogs”. As used herein a “derivative” of abinding molecule or fragment thereof, an antibody, or an antibodypolypeptide refers to a subject polypeptide having one or more residueschemically derivatized by reaction of a functional side group. Alsoincluded as “derivatives” are those peptides which contain one or morenaturally occurring amino acid derivatives of the twenty standard aminoacids. For example, 4-hydroxyproline may be substituted for proline;5-hydroxylysine may be substituted for lysine; 3-methylhistidine may besubstituted for histidine; homoserine may be substituted for serine; andornithine may be substituted for lysine.

The term “polynucleotide” is intended to encompass a singular nucleicacid as well as plural nucleic acids, and refers to an isolated nucleicacid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA(pDNA). A polynucleotide may comprise a conventional phosphodiester bondor a non-conventional bond (e.g., an amide bond, such as found inpeptide nucleic acids (PNA)). The term “nucleic acid” refers to any oneor more nucleic acid segments, e.g., DNA or RNA fragments, present in apolynucleotide. By “isolated” nucleic acid or polynucleotide is intendeda nucleic acid molecule, DNA or RNA, which has been removed from itsnative environment. For example, a recombinant polynucleotide encodingan antibody contained in a vector is considered isolated for thepurposes of the present invention. Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in solution. Isolated RNA molecules include in vivo orin vitro RNA transcripts of polynucleotides of the present invention.Isolated polynucleotides or nucleic acids according to the presentinvention further include such molecules produced synthetically. Inaddition, polynucleotide or a nucleic acid may be or may include aregulatory element such as a promoter, ribosome binding site, or atranscription terminator.

As used herein, a “coding region” is a portion of nucleic acid whichconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it may beconsidered to be part of a coding region, but any flanking sequences,for example promoters, ribosome binding sites, transcriptionalterminators, introns, and the like, are not part of a coding region. Twoor more coding regions of the present invention can be present in asingle polynucleotide construct, e.g., on a single vector, or inseparate polynucleotide constructs, e.g., on separate (different)vectors. Furthermore, any vector may contain a single coding region, ormay comprise two or more coding regions, e.g., a single vector mayseparately encode an immunoglobulin heavy chain variable region and animmunoglobulin light chain variable region. In addition, a vector,polynucleotide, or nucleic acid of the invention may encode heterologouscoding regions, either fused or unfused to a nucleic acid encoding abinding molecule, an antibody, or fragment, variant, or derivativethereof. Heterologous coding regions include without limitationspecialized elements or motifs, such as a secretory signal peptide or aheterologous functional domain.

In certain embodiments, the polynucleotide or nucleic acid is DNA. Inthe case of DNA, a polynucleotide comprising a nucleic acid whichencodes a polypeptide normally may include a promoter and/or othertranscription or translation control elements operably associated withone or more coding regions. An operable association is when a codingregion for a gene product, e.g., a polypeptide, is associated with oneor more regulatory sequences in such a way as to place expression of thegene product under the influence or control of the regulatorysequence(s). Two DNA fragments (such as a polypeptide coding region anda promoter associated therewith) are “operably associated” or “operablylinked” if induction of promoter function results in the transcriptionof mRNA encoding the desired gene product and if the nature of thelinkage between the two DNA fragments does not interfere with theability of the expression regulatory sequences to direct the expressionof the gene product or interfere with the ability of the DNA template tobe transcribed. Thus, a promoter region would be operably associatedwith a nucleic acid encoding a polypeptide if the promoter was capableof effecting transcription of that nucleic acid. The promoter may be acell-specific promoter that directs substantial transcription of the DNAonly in predetermined cells. Other transcription control elements,besides a promoter, for example enhancers, operators, repressors, andtranscription termination signals, can be operably associated with thepolynucleotide to direct cell-specific transcription. Suitable promotersand other transcription control regions are disclosed herein.

A variety of transcription control regions are known to those skilled inthe art. These include, without limitation, transcription controlregions which function in vertebrate cells, such as, but not limited to,promoter and enhancer segments from cytomegaloviruses (the immediateearly promoter, in conjunction with intron-A), simian virus 40 (theearly promoter), and retroviruses (such as Rous sarcoma virus). Othertranscription control regions include those derived from vertebrategenes such as actin, heat shock protein, bovine growth hormone andrabbit β-globin, as well as other sequences capable of controlling geneexpression in eukaryotic cells. Additional suitable transcriptioncontrol regions include tissue-specific promoters and enhancers as wellas lymphokine-inducible promoters (e.g., promoters inducible byinterferons or interleukins).

Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited toribosome binding sites, translation initiation and termination codons,and elements derived from picornaviruses (particularly an internalribosome entry site, or IRES, also referred to as a CITE sequence).

In other embodiments, a polynucleotide of the present invention is RNA,for example, in the form of messenger RNA (mRNA).

Polynucleotide and nucleic acid coding regions of the present inventionmay be associated with additional coding regions which encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. According to the signalhypothesis, proteins secreted by mammalian cells have a signal peptideor secretory leader sequence which is cleaved from the mature proteinonce export of the growing protein chain across the rough endoplasmicreticulum has been initiated. Those of ordinary skill in the art areaware that polypeptides secreted by vertebrate cells generally have asignal peptide fused to the N-terminus of the polypeptide, which iscleaved from the complete or “full length” polypeptide to produce asecreted or “mature” form of the polypeptide. In certain embodiments,the native signal peptide, e.g., an immunoglobulin heavy chain or lightchain signal peptide is used, or a functional derivative of thatsequence that retains the ability to direct the secretion of thepolypeptide that is operably associated with it. Alternatively, aheterologous mammalian signal peptide, or a functional derivativethereof, may be used. For example, the wild-type leader sequence may besubstituted with the leader sequence of human tissue plasminogenactivator (TPA) or mouse β-glucuronidase.

Unless stated otherwise, the terms “disorder” and “disease” are usedinterchangeably herein.

A “binding molecule” as used in the context of the present inventionrelates primarily to antibodies, and fragments thereof, but may alsorefer to other non-antibody molecules that bind to α-synuclein includingbut not limited to hormones, receptors, ligands, majorhistocompatibility complex (MHC) molecules, chaperones such as heatshock proteins (HSPs) as well as cell-cell adhesion molecules such asmembers of the cadherin, intergrin, C-type lectin and immunoglobulin(Ig) superfamilies. Thus, for the sake of clarity only and withoutrestricting the scope of the present invention most of the followingembodiments are discussed with respect to antibodies and antibody-likemolecules which represent the preferred binding molecules for thedevelopment of therapeutic and diagnostic agents.

The terms “antibody” and “immunoglobulin” are used interchangeablyherein. An antibody or immunoglobulin is an α-synuclein-binding moleculewhich comprises at least the variable domain of a heavy chain, andnormally comprises at least the variable domains of a heavy chain and alight chain. Basic immunoglobulin structures in vertebrate systems arerelatively well understood; see, e.g., Harlow et al., Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).

As will be discussed in more detail below, the term “immunoglobulin”comprises various broad classes of polypeptides that can bedistinguished biochemically. Those skilled in the art will appreciatethat heavy chains are classified as gamma, mu, alpha, delta, or epsilon,(γ, μ, α, δ, ϵ) with some subclasses among them (e.g., γ1-γ4). It is thenature of this chain that determines the “class” of the antibody as IgG,IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses(isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc. are wellcharacterized and are known to confer functional specialization.Modified versions of each of these classes and isotypes are readilydiscernable to the skilled artisan in view of the instant disclosureand, accordingly, are within the scope of the instant invention. Allimmunoglobulin classes are clearly within the scope of the presentinvention, the following discussion will generally be directed to theIgG class of immunoglobulin molecules. With regard to IgG, a standardimmunoglobulin molecule comprises two identical light chain polypeptidesof molecular weight approximately 23,000 Daltons, and two identicalheavy chain polypeptides of molecular weight 53,000-70,000. The fourchains are typically joined by disulfide bonds in a “Y” configurationwherein the light chains bracket the heavy chains starting at the mouthof the “Y” and continuing through the variable region.

Light chains are classified as either kappa or lambda (κ, λ). Each heavychain class may be bound with either a kappa or lambda light chain. Ingeneral, the light and heavy chains are covalently bonded to each other,and the “tail” portions of the two heavy chains are bonded to each otherby covalent disulfide linkages or non-covalent linkages when theimmunoglobulins are generated either by hybridomas, B cells orgenetically engineered host cells. In the heavy chain, the amino acidsequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain.

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the light (V_(L)) and heavy (V_(H)) chain portionsdetermine antigen recognition and specificity. Conversely, the constantdomains of the light chain (CL) and the heavy chain (CH1, CH2 or CH3)confer important biological properties such as secretion, transplacentalmobility, Fc receptor binding, complement binding, and the like. Byconvention the numbering of the constant region domains increases asthey become more distal from the antigen-binding site or amino-terminusof the antibody. The N-terminal portion is a variable region and at theC-terminal portion is a constant region; the CH3 and CL domains actuallycomprise the carboxy-terminus of the heavy and light chain,respectively.

As indicated above, the variable region allows the antibody toselectively recognize and specifically bind epitopes on antigens. Thatis, the V_(L) domain and V_(H) domain, or subset of the complementaritydetermining regions (CDRs), of an antibody combine to form the variableregion that defines a three dimensional antigen-binding site. Thisquaternary antibody structure forms the antigen-binding site present atthe end of each arm of the Y. More specifically, the antigen-bindingsite is defined by three CDRs on each of the V_(H) and V_(L) chains. Anyantibody or immunoglobulin fragment which contains sufficient structureto specifically bind to α-synuclein is denoted herein interchangeably asa “binding fragment” or an “immunospecific fragment.”

In naturally occurring antibodies, an antibody comprises sixhypervariable regions, sometimes called “complementarity determiningregions” or “CDRs” present in each antigen-binding domain, which areshort, non-contiguous sequences of amino acids that are specificallypositioned to form the antigen-binding domain as the antibody assumesits three dimensional configuration in an aqueous environment. The“CDRs” are flanked by four relatively conserved “framework” regions or“FRs” which show less inter-molecular variability. The framework regionslargely adopt a β-sheet conformation and the CDRs form loops whichconnect, and in some cases form part of, the β-sheet structure. Thus,framework regions act to form a scaffold that provides for positioningthe CDRs in correct orientation by inter-chain, non-covalentinteractions. The antigen-binding domain formed by the positioned CDRsdefines a surface complementary to the epitope on the immunoreactiveantigen. This complementary surface promotes the non-covalent binding ofthe antibody to its cognate epitope. The amino acids comprising the CDRsand the framework regions, respectively, can be readily identified forany given heavy or light chain variable region by one of ordinary skillin the art, since they have been precisely defined; see, “Sequences ofProteins of Immunological Interest,” Kabat, E., et al., U.S. Departmentof Health and Human Services, (1983); and Chothia and Lesk, J. Mol.Biol., 196 (1987), 901-917, which are incorporated herein by referencein their entireties.

In the case where there are two or more definitions of a term which isused and/or accepted within the art, the definition of the term as usedherein is intended to include all such meanings unless explicitly statedto the contrary. A specific example is the use of the term“complementarity determining region” (“CDR”) to describe thenon-contiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. This particular region hasbeen described by Kabat et al., U.S. Dept. of Health and Human Services,“Sequences of Proteins of Immunological Interest” (1983) and by Chothiaand Lesk, J. Mol. Biol., 196 (1987), 901-917, which are incorporatedherein by reference, where the definitions include overlapping orsubsets of amino acid residues when compared against each other.Nevertheless, application of either definition to refer to a CDR of anantibody or variants thereof is intended to be within the scope of theterm as defined and used herein. The appropriate amino acid residueswhich encompass the CDRs as defined by each of the above citedreferences are set forth below in Table 1 as a comparison. The exactresidue numbers which encompass a particular CDR will vary depending onthe sequence and size of the CDR. Those skilled in the art can routinelydetermine which residues comprise a particular hypervariable region orCDR of the human IgG subtype of antibody given the variable region aminoacid sequence of the antibody.

TABLE 1 CDR Definitions¹ Kabat Chothia VH CDR1 31-35 26-32 VH CDR2 50-6552-58 VH CDR3  95-102  95-102 VL CDR1 24-34 26-32 VL CDR2 50-56 50-52 VLCDR3 89-97 91-96 ¹Numbering of all CDR definitions in Table 1 isaccording to the numbering conventions set forth by Kabat et al. (seebelow).

Kabat et al. also defined a numbering system for variable domainsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable domain sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).Unless otherwise specified, references to the numbering of specificamino acid residue positions in an antibody or antigen-binding fragment,variant, or derivative thereof of the present invention are according tothe Kabat numbering system.

Antibodies or antigen-binding fragments, immunospecific fragments,variants, or derivatives thereof of the invention include, but are notlimited to, polyclonal, monoclonal, multispecific, human, humanized,primatized, murinized or chimeric antibodies, single chain antibodies,epitope-binding fragments, e.g., Fab, Fab′ and F(ab′)_(2,) Fd, Fvs,single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs(sdFv), fragments comprising either a V_(L) or V_(H) domain, fragmentsproduced by a Fab expression library, and anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies disclosedherein). ScFv molecules are known in the art and are described, e.g., inU.S. Pat. No. 5,892,019. Immunoglobulin or antibody molecules of theinvention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY),class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule.

In one embodiment, the antibody of the present invention is not IgM or aderivative thereof with a pentavalent structure. Particular, in specificapplications of the present invention, especially therapeutic use, IgMsare less useful than IgG and other bivalent antibodies or correspondingbinding molecules since IgMs due to their pentavalent structure and lackof affinity maturation often show unspecific cross-reactivities and verylow affinity.

In a particularly preferred embodiment, the antibody of the presentinvention is not a polyclonal antibody, i.e. it substantially consistsof one particular antibody species rather than being a mixture obtainedfrom a plasma immunoglobulin sample.

Antibody fragments, including single-chain antibodies, may comprise thevariable region(s) alone or in combination with the entirety or aportion of the following: hinge region, CH1, CH2, and CH3 domains. Alsoincluded in the invention are α-synuclein-binding fragments alsocomprising any combination of variable region(s) with a hinge region,CH1, CH2, and CH3 domains. Antibodies or immunospecific fragmentsthereof of the present invention may be from any animal origin includingbirds and mammals. Preferably, the antibodies are human, murine, donkey,rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. Inanother embodiment, the variable region may be condricthoid in origin(e.g., from sharks).

In one aspect, the antibody of the present invention is a humanmonoclonal antibody isolated from a human. Optionally, the frameworkregion of the human antibody is aligned and adopted in accordance withthe pertinent human germ line variable region sequences in the database;see, e.g., Vbase (http://vbase.mrc-cpe.cam.ac.uk/) hosted by the MRCCentre for Protein Engineering (Cambridge, UK). For example, amino acidsconsidered to potentially deviate from the true germ line sequence couldbe due to the PCR primer sequences incorporated during the cloningprocess. Compared to artificially generated human-like antibodies suchas single chain antibody fragments (scFvs) from a phage displayedantibody library or xenogeneic mice the human monoclonal antibody of thepresent invention is characterized by (i) being obtained using the humanimmune response rather than that of animal surrogates, i.e. the antibodyhas been generated in response to natural α-synuclein in its relevantconformation in the human body, (ii) having protected the individual oris at least significant for the presence of α-synuclein, and (iii) sincethe antibody is of human origin the risks of cross-reactivity againstself-antigens is minimized. Thus, in accordance with the presentinvention the terms “human monoclonal antibody”, “human monoclonalautoantibody”, “human antibody” and the like are used to denote anα-synuclein binding molecule which is of human origin, i.e. which hasbeen isolated from a human cell such as a B cell or hybridoma thereof orthe cDNA of which has been directly cloned from mRNA of a human cell,for example a human memory B cell. A human antibody is still “human”even if amino acid substitutions are made in the antibody, e.g., toimprove binding characteristics.

Antibodies derived from human immunoglobulin libraries or from animalstransgenic for one or more human immunoglobulins and that do not expressendogenous immunoglobulins, as described infra and, for example in, U.S.Pat. No. 5,939,598 by Kucherlapati et al., are denoted human-likeantibodies in order distinguish them from truly human antibodies of thepresent invention.

As used herein, the term “murinized antibody” or “murinizedimmunoglobulin” refers to an antibody comprising one or more CDRs from ahuman antibody of the present invention; and a human framework regionthat contains amino acid substitutions and/or deletions and/orinsertions that are based on a mouse antibody sequence. The humanimmunoglobulin providing the CDRs is called the “parent” or “acceptor”and the mouse antibody providing the framework changes is called the“donor”. Constant regions need not be present, but if they are, they areusually substantially identical to mouse antibody constant regions, i.e.at least about 85-90%, preferably about 95% or more identical. Hence, insome embodiments, a full length murinized human heavy or light chainimmunoglobulin contains a mouse constant region, human CDRs, and asubstantially human framework that has a number of “murinizing” aminoacid substitutions. Typically, a “murinized antibody” is an antibodycomprising a murinized variable light chain and/or a murinized variableheavy chain. For example, a murinized antibody would not encompass atypical chimeric antibody, e.g., because the entire variable region of achimeric antibody is non-mouse. A modified antibody that has been“murinized” by the process of “murinization” binds to the same antigenas the parent antibody that provides the CDRs and is usually lessimmunogenic in mice, as compared to the parent antibody.

As used herein, the term “heavy chain portion” includes amino acidsequences derived from an immunoglobulin heavy chain. A polypeptidecomprising a heavy chain portion comprises at least one of: a CH1domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain,a CH2 domain, a CH3 domain, or a variant or fragment thereof. Forexample, a binding polypeptide for use in the invention may comprise apolypeptide chain comprising a CH1 domain; a polypeptide chaincomprising a CH1 domain, at least a portion of a hinge domain, and a CH2domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; apolypeptide chain comprising a CH1 domain, at least a portion of a hingedomain, and a CH3 domain, or a polypeptide chain comprising a CH1domain, at least a portion of a hinge domain, a CH2 domain, and a CH3domain. In another embodiment, a polypeptide of the invention comprisesa polypeptide chain comprising a CH3 domain. Further, a bindingpolypeptide for use in the invention may lack at least a portion of aCH2 domain (e.g., all or part of a CH2 domain). As set forth above, itwill be understood by one of ordinary skill in the art that thesedomains (e.g., the heavy chain portions) may be modified such that theyvary in amino acid sequence from the naturally occurring immunoglobulinmolecule.

In certain antibodies, or antigen-binding fragments, variants, orderivatives thereof disclosed herein, the heavy chain portions of onepolypeptide chain of a multimer are identical to those on a secondpolypeptide chain of the multimer. Alternatively, heavy chainportion-containing monomers of the invention are not identical. Forexample, each monomer may comprise a different target binding site,forming, for example, a bispecific antibody or diabody.

In another embodiment, the antibodies, or antigen-binding fragments,variants, or derivatives thereof disclosed herein are composed of asingle polypeptide chain such as scFvs and are to be expressedintracellularly (intrabodies) for potential in vivo therapeutic anddiagnostic applications.

The heavy chain portions of a binding polypeptide for use in thediagnostic and treatment methods disclosed herein may be derived fromdifferent immunoglobulin molecules. For example, a heavy chain portionof a polypeptide may comprise a CH1 domain derived from an IgG1 moleculeand a hinge region derived from an IgG3 molecule. In another example, aheavy chain portion can comprise a hinge region derived, in part, froman IgG1 molecule and, in part, from an IgG3 molecule. In anotherexample, a heavy chain portion can comprise a chimeric hinge derived, inpart, from an IgG1 molecule and, in part, from an IgG4 molecule.

As used herein, the term “light chain portion” includes amino acidsequences derived from an immunoglobulin light chain. Preferably, thelight chain portion comprises at least one of a V_(L) or CL domain.

The minimum size of a peptide or polypeptide epitope for an antibody isthought to be about four to five amino acids. Peptide or polypeptideepitopes preferably contain at least seven, more preferably at leastnine and most preferably between at least about 15 to about 30 aminoacids. Since a CDR can recognize an antigenic peptide or polypeptide inits tertiary form, the amino acids comprising an epitope need not becontiguous, and in some cases, may not even be on the same peptidechain. In the present invention, a peptide or polypeptide epitoperecognized by antibodies of the present invention contains a sequence ofat least 4, at least 5, at least 6, at least 7, more preferably at least8, at least 9, at least 10, at least 15, at least 20, at least 25, orbetween about 15 to about 30 contiguous or non-contiguous amino acids ofα-synuclein.

By “specifically binding”, or “specifically recognizing”, usedinterchangeably herein, it is generally meant that a binding molecule,e.g., an antibody binds to an epitope via its antigen-binding domain,and that the binding entails some complementarity between theantigen-binding domain and the epitope. According to this definition, anantibody is said to “specifically bind” to an epitope when it binds tothat epitope, via its antigen-binding domain more readily than it wouldbind to a random, unrelated epitope. The term “specificity” is usedherein to qualify the relative affinity by which a certain antibodybinds to a certain epitope. For example, antibody “A” may be deemed tohave a higher specificity for a given epitope than antibody “B,” orantibody “A” may be said to bind to epitope “C” with a higherspecificity than it has for related epitope “D”.

Where present, the term “immunological binding characteristics,” orother binding characteristics of an antibody with an antigen, in all ofits grammatical forms, refers to the specificity, affinity,cross-reactivity, and other binding characteristics of an antibody.

By “preferentially binding”, it is meant that the binding molecule,e.g., antibody specifically binds to an epitope more readily than itwould bind to a related, similar, homologous, or analogous epitope.Thus, an antibody which “preferentially binds” to a given epitope wouldmore likely bind to that epitope than to a related epitope, even thoughsuch an antibody may cross-react with the related epitope.

By way of non-limiting example, a binding molecule, e.g., an antibodymay be considered to bind a first epitope preferentially if it bindssaid first epitope with a dissociation constant (K_(D)) that is lessthan the antibody's K_(D) for the second epitope. In anothernon-limiting example, an antibody may be considered to bind a firstantigen preferentially if it binds the first epitope with an affinitythat is at least one order of magnitude less than the antibody's K_(D)for the second epitope. In another non-limiting example, an antibody maybe considered to bind a first epitope preferentially if it binds thefirst epitope with an affinity that is at least two orders of magnitudeless than the antibody's K_(D) for the second epitope.

In another non-limiting example, a binding molecule, e.g., an antibodymay be considered to bind a first epitope preferentially if it binds thefirst epitope with an off rate (k(off)) that is less than the antibody'sk(off) for the second epitope. In another non-limiting example, anantibody may be considered to bind a first epitope preferentially if itbinds the first epitope with an affinity that is at least one order ofmagnitude less than the antibody's k(off) for the second epitope. Inanother non-limiting example, an antibody may be considered to bind afirst epitope preferentially if it binds the first epitope with anaffinity that is at least two orders of magnitude less than theantibody's k(off) for the second epitope.

A binding molecule, e.g., an antibody or antigen-binding fragment,variant, or derivative disclosed herein may be said to bind aα-synuclein or a fragment or variant thereof with an off rate (k(off))of less than or equal to 5×10⁻² sec⁻¹, 10⁻² sec⁻¹, 5×10⁻³ sec⁻¹ or 10⁻³sec⁻¹. More preferably, an antibody of the invention may be said to bindα-synuclein or a fragment or variant thereof with an off rate (k(off))less than or equal to 5×10⁻⁴ sec⁻¹, 10⁻⁴ sec⁻¹, 5×10⁻⁵ sec⁻¹, or 10⁻⁵sec⁻¹ 5×10⁶ sec⁻¹, 10⁻⁶ sec⁻¹, 5×10⁻⁷ sec⁻¹ or 10⁻⁷ sec⁻¹.

A binding molecule, e.g., an antibody or antigen-binding fragment,variant, or derivative disclosed herein may be said to bind α-synucleinor a fragment or variant thereof with an on rate (k(on)) of greater thanor equal to 10³ M³¹ ¹ sec⁻¹, 5×10³ M⁻¹ sec⁻¹, 10⁴ M⁻¹ sec⁻¹ or 5×10⁴ M⁻¹sec⁻¹. More preferably, an antibody of the invention may be said to bindα-synuclein or a fragment or variant thereof with an on rate (k(on))greater than or equal to 10⁵ M⁻¹ sec⁻¹, 5×10⁵ M⁻¹ sec⁻¹, 10⁶ M⁻¹ sec⁻¹,or 5×10⁶ M⁻¹ sec⁻¹ or 10⁷ M⁻¹ sec⁻¹.

A binding molecule, e.g., an antibody is said to competitively inhibitbinding of a reference antibody to a given epitope if it preferentiallybinds to that epitope to the extent that it blocks, to some degree,binding of the reference antibody to the epitope. Competitive inhibitionmay be determined by any method known in the art, for example,competition ELISA assays. An antibody may be said to competitivelyinhibit binding of the reference antibody to a given epitope by at least90%, at least 80%, at least 70%, at least 60%, or at least 50%.

As used herein, the term “affinity” refers to a measure of the strengthof the binding of an individual epitope with the CDR of a bindingmolecule, e.g., an immunoglobulin molecule; see, e.g., Harlow et al.,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,2nd ed. (1988) at pages 27-28. As used herein, the term “avidity” refersto the overall stability of the complex between a population ofimmunoglobulins and an antigen, that is, the functional combiningstrength of an immunoglobulin mixture with the antigen; see, e.g.,Harlow at pages 29-34. Avidity is related to both the affinity ofindividual immunoglobulin molecules in the population with specificepitopes, and also the valencies of the immunoglobulins and the antigen.For example, the interaction between a bivalent monoclonal antibody andan antigen with a highly repeating epitope structure, such as a polymer,would be one of high avidity. The affinity or avidity of an antibody foran antigen can be determined experimentally using any suitable method;see, for example, Berzofsky et al., “Antibody-Antigen Interactions” InFundamental Immunology, Paul, W. E., Ed., Raven Press New York, N.Y.(1984), Kuby, Janis Immunology, W. H. Freeman and Company New York, N.Y.(1992), and methods described herein. General techniques for measuringthe affinity of an antibody for an antigen include ELISA, RIA, andsurface plasmon resonance. The measured affinity of a particularantibody-antigen interaction can vary if measured under differentconditions, e.g., salt concentration, pH. Thus, measurements of affinityand other antigen-binding parameters, e.g., K_(D), IC_(50,) arepreferably made with standardized solutions of antibody and antigen, anda standardized buffer.

Binding molecules, e.g., antibodies or antigen-binding fragments,variants or derivatives thereof of the invention may also be describedor specified in terms of their cross-reactivity.

As used herein, the term “cross-reactivity” refers to the ability of anantibody, specific for one antigen, to react with a second antigen; ameasure of relatedness between two different antigenic substances. Thus,an antibody is cross reactive if it binds to an epitope other than theone that induced its formation. The cross reactive epitope generallycontains many of the same complementary structural features as theinducing epitope, and in some cases, may actually fit better than theoriginal.

For example, certain antibodies have some degree of cross-reactivity, inthat they bind related, but non-identical epitopes, e.g., epitopes withat least 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 65%, at least 60%, at least 55%, and at least 50%identity (as calculated using methods known in the art and describedherein) to a reference epitope. An antibody may be said to have littleor no cross-reactivity if it does not bind epitopes with less than 95%,less than 90%, less than 85%, less than 80%, less than 75%, less than70%, less than 65%, less than 60%, less than 55%, and less than 50%identity (as calculated using methods known in the art and describedherein) to a reference epitope. An antibody may be deemed “highlyspecific” for a certain epitope, if it does not bind any other analog,ortholog, or homolog of that epitope.

Binding molecules, e.g., antibodies or antigen-binding fragments,variants or derivatives thereof of the invention may also be describedor specified in terms of their binding affinity to α-synuclein.Preferred binding affinities include those with a dissociation constantor Kd less than 5×10⁻²M, 10⁻²M, 5×10⁻³M, 10⁻³M, 5×10⁻⁴M, 10⁻⁴M, 5×10⁻⁵M,10⁻⁵M, 5×10⁻⁶M, 10⁻⁶M, 5×10⁻⁷M, 10⁻⁷M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M,5×10⁻¹° M, 10⁻¹° M, 5 x 10⁻10⁻¹¹M, 5×10⁻¹²M, 10⁻¹²M, 5×10⁻¹³M, 10⁻¹³M,5×10⁻¹⁴M, 10⁻¹⁴M, 5×10⁻¹⁵M, or 10⁻¹⁵M.

As previously indicated, the subunit structures and three dimensionalconfiguration of the constant regions of the various immunoglobulinclasses are well known. As used herein, the term “V_(H) domain” includesthe amino terminal variable domain of an immunoglobulin heavy chain andthe term “CH1 domain” includes the first (most amino terminal) constantregion domain of an immunoglobulin heavy chain. The CH1 domain isadjacent to the V_(H) domain and is amino terminal to the hinge regionof an immunoglobulin heavy chain molecule.

As used herein the term “CH2 domain” includes the portion of a heavychain molecule that extends, e.g., from about residue 244 to residue 360of an antibody using conventional numbering schemes (residues 244 to360, Kabat numbering system; and residues 231-340, EU numbering system;see Kabat EA et al. op. cit). The CH2 domain is unique in that it is notclosely paired with another domain. Rather, two N-linked branchedcarbohydrate chains are interposed between the two CH2 domains of anintact native IgG molecule. It is also well documented that the CH3domain extends from the CH2 domain to the C-terminal of the IgG moleculeand comprises approximately 108 residues.

As used herein, the term “hinge region” includes the portion of a heavychain molecule that joins the CH1 domain to the CH2 domain. This hingeregion comprises approximately 25 residues and is flexible, thusallowing the two N-terminal antigen-binding regions to moveindependently. Hinge regions can be subdivided into three distinctdomains: upper, middle, and lower hinge domains; see Roux et al., J.Immunol. 161 (1998), 4083.

As used herein the term “disulfide bond” includes the covalent bondformed between two sulfur atoms. The amino acid cysteine comprises athiol group that can form a disulfide bond or bridge with a second thiolgroup. In most naturally occurring IgG molecules, the CH1 and CL regionsare linked by a disulfide bond and the two heavy chains are linked bytwo disulfide bonds at positions corresponding to 239 and 242 using theKabat numbering system (position 226 or 229, EU numbering system).

As used herein, the terms “linked,” “fused” or “fusion” are usedinterchangeably. These terms refer to the joining together of two moreelements or components, by whatever means including chemical conjugationor recombinant means. An “in-frame fusion” refers to the joining of twoor more polynucleotide open reading frames (ORFs) to form a continuouslonger ORF, in a manner that maintains the correct translational readingframe of the original ORFs. Thus, a recombinant fusion protein is asingle protein containing two or more segments that correspond topolypeptides encoded by the original ORFs (which segments are notnormally so joined in nature). Although the reading frame is thus madecontinuous throughout the fused segments, the segments may be physicallyor spatially separated by, for example, in-frame linker sequence. Forexample, polynucleotides encoding the CDRs of an immunoglobulin variableregion may be fused, in-frame, but be separated by a polynucleotideencoding at least one immunoglobulin framework region or additional CDRregions, as long as the “fused” CDRs are co-translated as part of acontinuous polypeptide.

The term “expression” as used herein refers to a process by which a geneproduces a biochemical, for example, an RNA or polypeptide. The processincludes any manifestation of the functional presence of the gene withinthe cell including, without limitation, gene knockdown as well as bothtransient expression and stable expression. It includes withoutlimitation transcription of the gene into messenger RNA (mRNA), transferRNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) orany other RNA product, and the translation of such mRNA intopolypeptide(s). If the final desired product is a biochemical,expression includes the creation of that biochemical and any precursors.Expression of a gene produces a “gene product.” As used herein, a geneproduct can be either a nucleic acid, e.g., a messenger RNA produced bytranscription of a gene, or a polypeptide which is translated from atranscript. Gene products described herein further include nucleic acidswith post transcriptional modifications, e.g., polyadenylation, orpolypeptides with post translational modifications, e.g., methylation,glycosylation, the addition of lipids, association with other proteinsubunits, proteolytic cleavage, and the like.

As used herein, the term “sample” refers to any biological materialobtained from a subject or patient. In one aspect, a sample can compriseblood, cerebrospinal fluid (“CSF”), or urine. In other aspects, a samplecan comprise whole blood, plasma, B cells enriched from blood samples,and cultured cells (e.g., B cells from a subject). A sample can alsoinclude a biopsy or tissue sample including neural tissue. In stillother aspects, a sample can comprise whole cells and/or a lysate of thecells. Blood samples can be collected by methods known in the art. Inone aspect, the pellet can be resuspended by vortexing at 4° C. in 200μl buffer (20 mM Tris, pH. 7.5, 0.5% Nonidet, 1 mM EDTA, 1 mM PMSF, 0.1MNaCl, IX Sigma Protease Inhibitor, and IX Sigma Phosphatase Inhibitors 1and 2). The suspension can be kept on ice for 20 minutes withintermittent vortexing. After spinning at 15,000 ×g for 5 minutes atabout 4° C., aliquots of supernatant can be stored at about −70° C.

As used herein, the terms “treat” or “treatment” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder, such as the development ofParkinsonism. Beneficial or desired clinical results include, but arenot limited to, alleviation of symptoms, diminishment of extent ofdisease, stabilized (i.e., not worsening) state of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.Those in need of treatment include those already with the condition ordisorder as well as those prone to have the condition or disorder orthose in which the manifestation of the condition or disorder is to beprevented.

By “subject” or “individual” or “animal” or “patient” or “mammal,” ismeant any subject, particularly a mammalian subject, e.g., a humanpatient, for whom diagnosis, prognosis, prevention, or therapy isdesired.

II. Antibodies

The present invention generally relates to human anti-α-synucleinantibodies and antigen-binding fragments thereof, which preferablydemonstrate the immunological binding characteristics and/or biologicalproperties as outlined for the antibodies illustrated in the Examples.In accordance with the present invention human monoclonal antibodiesspecific for α-synuclein were cloned from a pool of aged subjects.

In the course of the experiments performed in accordance with thepresent invention initial attempts failed to clone α-synuclein specificantibodies but almost always resulted in false-positive clones. Furtherinvestigation of these clones revealed that they produced antibodiesrecognizing proteins of E. coli. In order to circumvent this problem,antibodies in conditioned media of human memory B cell cultures werescreened in parallel for binding to coated full-length alpha synucleinmonomer and absence of binding to E. coli. proteins and bovine serumalbumin (BSA). In particular, B cell conditioned medium was preabsorbedwith E. coli proteins prior to subjecting the medium to an ELISA assayfor screening of α-synuclein binding human antibodies.

Initial attempts to isolating specific antibodies were focused at poolsof human subjects with high plasma binding activity to α-synuclein,suggestive of elevated levels of circulating α-synuclein antibodiesplasma. Unexpectedly, these attempts failed to produce α-synucleinspecific human memory B cells and the antibodies described in thecurrent invention were isolated from pools of subjects with low plasmareactivity to α-synuclein.

Due to this measure, several antibodies could be isolated. Selectedantibodies were further analyzed for class and light chain subclassdetermination. Selected relevant antibody messages from memory B cellcultures are then transcribed by RT-PCR, cloned and combined intoexpression vectors for recombinant production; see the appendedExamples. Recombinant expression of the human antibodies in HEK293 orCHO cells and the subsequent characterization of their bindingspecificities towards full-length α-synuclein and truncated formsthereof (FIG. 2), on Western Blot (FIG. 4) as well as to (β- andγ-synuclein (FIG. 3) confirmed that for the first time human antibodieshave been cloned that are highly specific for α-synuclein and recognizedifferent epitopes within the α-synuclein protein.

Thus, the present invention generally relates to an isolated naturallyoccurring human monoclonal anti-α-synuclein antibody and bindingfragments, derivatives and variants thereof. As demonstrated in theExamples and shown in FIG. 3 the human monoclonal anti-α-synucleinantibody of the present invention is preferably characterized inspecifically binding α-synuclein compared to β-synuclein andγ-synuclein. Advantageously, the antibody is capable of specificallybinding α-synuclein in the native monomer form and/or in the oligomericor aggregated form. In addition, the human anti-α-synuclein antibody ofthe present invention may be further characterized by its ability torecognize α-synuclein on Western Blotting; see FIG. 4.

In one embodiment, the present invention is directed to ananti-α-synuclein antibody, or antigen-binding fragment, variant orderivatives thereof, where the antibody specifically binds to the sameepitope of α-synuclein as a reference antibody selected from the groupconsisting of NI-202.3G12, NI-202.12F4 or NI-202.3D8. As illustrated inthe Examples, antibody NI-202.3G12 binds wild type (wt) α-synuclein butnot to α-synuclein truncations in a direct ELISA assay, pointing to astructural epitope of NI-202.3G12; see FIG. 2A. In contrast, antibodyNI-202.12F4 binds to α-synuclein truncations containing the N-terminalamphipathic repeat region (amino acids 1-60) in a direct ELISA assay,pointing to an N-terminal epitope of NI-202.12F4; see FIG. 2B. Inaddition, preliminary results of direct ELISA assays performed withantibody NI-202.3D8 revealed that NI-202.3D8 specifically recognizes theC-terminus of α-synuclein, preferably amino acids 96-140.

Furthermore, without intending to be bound by initial experimentalobservations further preliminary experiments give rise to assume thatantibody NI-202.3D8 preferentially binds to α-synuclein monomer ratherthan fibrils in a direct ELISA antibody assay while antibodiesNI-202.12F4 and NI-202.3G12 preferentially bind α-synuclein aggregatesor fibrils over the monomeric form of α-synuclein. Hence, the presentinvention provides a set of human anti-α-synuclein antibodies withdifferent specificities, which are thus particularly useful fordiagnostic and therapeutic purposes.

In one embodiment, the antibody of the present invention exhibits thebinding properties of the exemplary NI-202.12F4 antibody as described inany one of Examples 1 to 5. For example, in one embodiment theanti-α-synuclein antibody of the present invention preferentiallyrecognizes human rather than mouse α-synuclein, in particular whenanalyzed according to Example 3. In addition, or alternatively, theanti-α-synuclein antibody of the present invention preferentiallyrecognizes aggregated or misfolded forms α-synuclein rather thanphysiological monomeric forms, in particular when analyzed according toExample 3. In addition, or alternatively, the anti-α-synuclein antibodyof the present invention binds to disease causing mutants of humanα-synuclein, in particular those described in Example 3. In thiscontext, the binding specificities may be in the range as shown for theexemplary NI-202.12F4 antibody in FIG. 5, i.e. having half maximaleffective concentrations (EC50) of about 100 to 1000 pM, preferably anEC50 of about 100 to 500 pM for wild-type α-synuclein or a diseasecausing mutant thereof.

Hence, the anti-α-synuclein antibody of the present invention preferablypreferentially binds to pathological forms of α-synuclein in brain, e.g.pathological aggregates of α-synuclein as exemplified by Western blotand immunohistochemical staining described in Example 3. Accordingly, inanother additional or alternative embodiment the anti-α-synucleinantibody of the present invention preferentially binds to aconformational epitope of human α-synuclein and does not significantlybind N-terminal derived fragments of α-synuclein consisting of aminoacids 1-20; 21-40; 41-60; 11-30; or 31-50.

The present invention is also drawn to an antibody, or antigen-bindingfragment, variant or derivatives thereof, where the antibody comprisesan antigen-binding domain identical to that of an antibody selected fromthe group consisting of NI-202.3G12, NI-202.12F4 or NI-202.3D8.

The present invention further exemplifies several such bindingmolecules, e.g. antibodies and binding fragments thereof, which may becharacterized by comprising in their variable region, e.g. bindingdomain at least one complementarity determining region (CDR) of theV_(H) and/or V_(L) variable region comprising any one of the amino acidsequences depicted in FIG. 1. The corresponding nucleotide sequencesencoding the above-identified variable regions are set forth in theattached sequence listing. An exemplary set of CDRs of the above aminoacid sequences of the V_(H) and/or V_(L) region as depicted in FIG. 1 isalso indicated in the appended sequence listing. However, as discussedin the following the person skilled in the art is well aware of the factthat in addition or alternatively CDRs may be used, which differ intheir amino acid sequence from those set forth in FIG. 1 by one, two,three or even more amino acids in case of CDR2 and CDR3.

In one embodiment, the antibody of the present invention is any one ofthe antibodies comprising an amino acid sequence of the V_(H) and/orV_(L) region as depicted in FIG. 1. Alternatively, the antibody of thepresent invention is an antibody or antigen-binding fragment, derivativeor variant thereof, which competes for binding to α-synuclein with atleast one of the antibodies having the V_(H) and/or V_(L) region asdepicted in FIG. 1. Those antibodies may be human as well, in particularfor therapeutic applications. Alternatively, the antibody is a murine,murinized and chimeric murine-human antibody, which are particularlyuseful for diagnostic methods and studies in animals.

As mentioned above, due to its generation upon a human immune responsethe human monoclonal antibody of the present invention will recognizeepitopes which are of particular physiological relevance and which mightnot be accessible or less immunogenic in case of immunization processesfor the generation of for example mouse monoclonal antibodies and in invitro screening of phage display libraries, respectively. Accordingly,it is prudent to stipulate that the epitope of the humananti-α-synuclein antibody of the present invention is unique and noother antibody which is capable of binding to the epitope recognized bythe human monoclonal antibody of the present invention exists.Therefore, the present invention also extends generally toanti-α-synuclein antibodies and α-synuclein binding molecules whichcompete with the human monoclonal antibody of the present invention forspecific binding to α-synuclein. The present invention is morespecifically directed to an antibody, or antigen-binding fragment,variant or derivatives thereof, where the antibody specifically binds tothe same epitope of α-synuclein as a reference antibody selected fromthe group consisting of NI-202.3G12, NI-202.12F4 or NI-202.3D8.

Competition between antibodies is determined by an assay in which theimmunoglobulin under test inhibits specific binding of a referenceantibody to a common antigen, such as α-synuclein. Numerous types ofcompetitive binding assays are known, for example: solid phase direct orindirect radioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay; see Stahli et al.,Methods in Enzymology 9 (1983), 242-253; solid phase directbiotin-avidin EIA; see Kirkland et al., J. Immunol. 137 (1986),3614-3619 and Cheung et al., Virology 176 (1990), 546-552; solid phasedirect labeled assay, solid phase direct labeled sandwich assay; seeHarlow and Lane, Antibodies, A Laboratory Manual, Cold Spring HarborPress (1988); solid phase direct label RIA using I¹²⁵ label; see Morelet al, Molec. Immunol. 25 (1988), 7-15 and Moldenhauer et al., Scand. J.Immunol. 32 (1990), 77-82. Typically, such an assay involves the use ofpurified α-synuclein or aggregates thereof bound to a solid surface orcells bearing either of these, an unlabelled test immunoglobulin and alabeled reference immunoglobulin, i.e. the human monoclonal antibody ofthe present invention. Competitive inhibition is measured by determiningthe amount of label bound to the solid surface or cells in the presenceof the test immunoglobulin. Usually the test immunoglobulin is presentin excess. Preferably, the competitive binding assay is performed underconditions as described for the ELISA assay in the appended Examples.Antibodies identified by competition assay (competing antibodies)include antibodies binding to the same epitope as the reference antibodyand antibodies binding to an adjacent epitope sufficiently proximal tothe epitope bound by the reference antibody for steric hindrance tooccur. Usually, when a competing antibody is present in excess, it willinhibit specific binding of a reference antibody to a common antigen byat least 50% or 75%. Hence, the present invention is further drawn to anantibody, or antigen-binding fragment, variant or derivatives thereof,where the antibody competitively inhibits a reference antibody selectedfrom the group consisting of NI-202.3G12, NI-202.12F4 or NI-202.3D8 frombinding to α-synuclein.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (V_(H)), where at least oneof V_(H)-CDRs of the heavy chain variable region or at least two of theV_(H)-CDRs of the heavy chain variable region are at least 80%, 85%, 90%or 95% identical to reference heavy chain V_(H)-CDR1, V_(H)-CDR2 orV_(H)-CDR3 amino acid sequences from the antibodies disclosed herein.Alternatively, the V_(H)-CDR1, V_(H)-CDR2 and V_(H)-CDR3 regions of theV_(H) are at least 80%, 85%, 90% or 95% identical to reference heavychain V_(H) -CDR1, V_(H)-CDR2 and V_(H)-CDR3 amino acid sequences fromthe antibodies disclosed herein. Thus, according to this embodiment aheavy chain variable region of the invention has V_(H)-CDR1, V_(H)-CDR2and V_(H)-CDR3 polypeptide sequences related to the groups shown inFIG. 1. While FIG. 1 shows V_(H)-CDRs defined by the Kabat system, otherCDR definitions, e.g., V_(H)-CDRs defined by the Chothia system, arealso included in the present invention, and can be easily identified bya person of ordinary skill in the art using the data presented in FIG.1.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (V_(H)) in which theV_(H)-CDR1, V_(H)-CDR2 and V_(H)-CDR3 regions have polypeptide sequenceswhich are identical to the V_(H)-CDR1, V_(H)-CDR2 and V_(H)-CDR3 groupsshown in FIG. 1.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (V_(H)) in which theV_(H)-CDR1, V_(H)-CDR2 and V_(H)-CDR3 regions have polypeptide sequenceswhich are identical to the V_(H)-CDR1, V_(H)-CDR2 and V_(H)-CDR3 groupsshown in FIG. 1, except for one, two, three, four, five, or six aminoacid substitutions in any one V_(H)-CDR. In certain embodiments theamino acid substitutions are conservative.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin light chain variable region (V_(L)), where at least oneof the V_(L)-CDRs of the light chain variable region or at least two ofthe V_(L)-CDRs of the light chain variable region are at least 80%, 85%,90% or 95% identical to reference light chain V_(L)-CDR1, V_(L)-CDR2 orV_(L)-CDR3 amino acid sequences from antibodies disclosed herein.Alternatively, the V_(L)-CDR1, V_(L)-CDR2 and V_(L)-CDR3 regions of theV_(L) are at least 80%, 85%, 90% or 95% identical to reference lightchain V_(L)-CDR1, V_(L)-CDR2 and V_(L)-CDR3 amino acid sequences fromantibodies disclosed herein. Thus, according to this embodiment a lightchain variable region of the invention has V_(L)-CDR1, V_(L)-CDR2 andV_(L)-CDR3 polypeptide sequences related to the polypeptides shown inFIG. 1. While FIG.

1 shows V_(L)-CDRs defined by the Kabat system, other CDR definitions,e.g., V_(L)-CDRs defined by the Chothia system, are also included in thepresent invention.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin light chain variable region (V_(L)) in which theV_(L)-CDR1, V_(L)-CDR2 and V_(L)-CDR3 regions have polypeptide sequenceswhich are identical to the V_(L)-CDR1, V_(L)-CDR2 and V_(L)-CDR3 groupsshown in FIG. 1.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (V_(L)) in which theV_(L)-CDR1, V_(L)-CDR2 and V_(L)-CDR3 regions have polypeptide sequenceswhich are identical to the V_(L)-CDR1, V_(L)-CDR2 and V_(L)-CDR3 groupsshown in FIG. 1, except for one, two, three, four, five, or six aminoacid substitutions in any one V_(L)-CDR. In certain embodiments theamino acid substitutions are conservative.

An immunoglobulin or its encoding cDNA may be further modified. Thus, ina further embodiment the method of the present invention comprises anyone of the step(s) of producing a chimeric antibody, murinized antibody,single-chain antibody, Fab-fragment, bi-specific antibody, fusionantibody, labeled antibody or an analog of any one of those.Corresponding methods are known to the person skilled in the art and aredescribed, e.g., in Harlow and Lane “Antibodies, A Laboratory Manual”,CSH Press, Cold Spring Harbor (1988). When derivatives of saidantibodies are obtained by the phage display technique, surface plasmonresonance as employed in the BlAcore system can be used to increase theefficiency of phage antibodies which bind to the same epitope as that ofany one of the antibodies described herein (Schier, Human AntibodiesHybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995),7-13). The production of chimeric antibodies is described, for example,in international application WO89/09622. Methods for the production ofhumanized antibodies are described in, e.g., European application EP-A10 239 400 and international application WO90/07861. A further source ofantibodies to be utilized in accordance with the present invention areso-called xenogeneic antibodies. The general principle for theproduction of xenogeneic antibodies such as human-like antibodies inmice is described in, e.g., international applications WO91/10741,WO94/02602, WO96/34096 and WO 96/33735. As discussed above, the antibodyof the invention may exist in a variety of forms besides completeantibodies; including, for example, Fv, Fab and F(ab)_(2,) as well as insingle chains; see e.g. international application WO88/09344.

The antibodies of the present invention or their correspondingimmunoglobulin chain(s) can be further modified using conventionaltechniques known in the art, for example, by using amino aciddeletion(s), insertion(s), substitution(s), addition(s), and/orrecombination(s) and/or any other modification(s) known in the arteither alone or in combination. Methods for introducing suchmodifications in the DNA sequence underlying the amino acid sequence ofan immunoglobulin chain are well known to the person skilled in the art;see, e.g., Sambrook, Molecular Cloning A Laboratory Manual, Cold SpringHarbor Laboratory (1989) N.Y. and Ausubel, Current Protocols inMolecular Biology, Green Publishing Associates and Wiley Interscience,N.Y. (1994). Modifications of the antibody of the invention includechemical and/or enzymatic derivatizations at one or more constituentamino acids, including side chain modifications, backbone modifications,and N- and C-terminal modifications including acetylation,hydroxylation, methylation, amidation, and the attachment ofcarbohydrate or lipid moieties, cofactors, and the like. Likewise, thepresent invention encompasses the production of chimeric proteins whichcomprise the described antibody or some fragment thereof at the aminoterminus fused to heterologous molecule such as an immunostimulatoryligand at the carboxyl terminus; see, e.g., international applicationWO00/30680 for corresponding technical details.

Additionally, the present invention encompasses peptides including thosecontaining a binding molecule as described above, for example containingthe CDR3 region of the variable region of any one of the mentionedantibodies, in particular CDR3 of the heavy chain since it hasfrequently been observed that heavy chain CDR3 (HCDR3) is the regionhaving a greater degree of variability and a predominant participationin antigen-antibody interaction. Such peptides may easily be synthesizedor produced by recombinant means to produce a binding agent usefulaccording to the invention. Such methods are well known to those ofordinary skill in the art. Peptides can be synthesized for example,using automated peptide synthesizers which are commercially available.The peptides can also be produced by recombinant techniques byincorporating the DNA expressing the peptide into an expression vectorand transforming cells with the expression vector to produce thepeptide.

Hence, the present invention relates to any binding molecule, e.g., anantibody or binding fragment thereof which is oriented towards the humananti-α-synuclein antibodies of the present invention and display thementioned properties, i.e. which specifically recognize α-synuclein.Such antibodies and binding molecules can be tested for their bindingspecificity and affinity by ELISA and Western Blot andimmunohistochemisty as described herein, see, e.g., the Examples.Furthermore, preliminary results of subsequent experiments performed inaccordance with the present invention revealed that the humanant-α-synuclein antibody of the present invention, in particularantibody NI-202.12F4 recognizes α-synuclein inclusion bodies present onhuman brain sections of patients who suffered from dementia with Lewybodies (DLB) or Parkinson's disease (PD). Thus, in a particularpreferred embodiment of the present invention, the human antibody orbinding fragment, derivative or variant thereof recognizes α-synucleinon human DLB or PD brain sections.

As an alternative to obtaining immunoglobulins directly from the cultureof immortalized B cells or B memory cells, the immortalized cells can beused as a source of rearranged heavy chain and light chain loci forsubsequent expression and/or genetic manipulation. Rearranged antibodygenes can be reverse transcribed from appropriate mRNAs to produce cDNA.If desired, the heavy chain constant region can be exchanged for that ofa different isotype or eliminated altogether. The variable regions canbe linked to encode single chain Fv regions. Multiple Fv regions can belinked to confer binding ability to more than one target or chimericheavy and light chain combinations can be employed. Once the geneticmaterial is available, design of analogs as described above which retainboth their ability to bind the desired target is straightforward.Methods for the cloning of antibody variable regions and generation ofrecombinant antibodies are known to the person skilled in the art andare described, for example, Gilliland et al., Tissue Antigens 47 (1996),1-20; Doenecke et al., Leukemia 11 (1997), 1787-1792.

Once the appropriate genetic material is obtained and, if desired,modified to encode an analog, the coding sequences, including those thatencode, at a minimum, the variable regions of the heavy and light chain,can be inserted into expression systems contained on vectors which canbe transfected into standard recombinant host cells. A variety of suchhost cells may be used; for efficient processing, however, mammaliancells are preferred. Typical mammalian cell lines useful for thispurpose include, but are not limited to, CHO cells, HEK 293 cells, orNSO cells.

The production of the antibody or analog is then undertaken by culturingthe modified recombinant host under culture conditions appropriate forthe growth of the host cells and the expression of the coding sequences.The antibodies are then recovered by isolating them from the culture.The expression systems are preferably designed to include signalpeptides so that the resulting antibodies are secreted into the medium;however, intracellular production is also possible.

In accordance with the above, the present invention also relates to apolynucleotide encoding the antibody or equivalent binding molecule ofthe present invention, in case of the antibody preferably at least avariable region of an immunoglobulin chain of the antibody describedabove. Typically, said variable region encoded by the polynucleotidecomprises at least one complementarity determining region (CDR) of theV_(H) and/or V_(L) of the variable region of the said antibody.

The person skilled in the art will readily appreciate that the variabledomain of the antibody having the above-described variable domain can beused for the construction of other polypeptides or antibodies of desiredspecificity and biological function. Thus, the present invention alsoencompasses polypeptides and antibodies comprising at least one CDR ofthe above-described variable domain and which advantageously havesubstantially the same or similar binding properties as the antibodydescribed in the appended examples. The person skilled in the art knowsthat binding affinity may be enhanced by making amino acid substitutionswithin the CDRs or within the hypervariable loops (Chothia and Lesk, J.Mol. Biol. 196 (1987), 901-917) which partially overlap with the CDRs asdefined by Kabat; see, e.g., Riechmann, et al, Nature 332 (1988),323-327. Thus, the present invention also relates to antibodies whereinone or more of the mentioned CDRs comprise one or more, preferably notmore than two amino acid substitutions. Preferably, the antibody of theinvention comprises in one or both of its immunoglobulin chains two orall three CDRs of the variable regions as set forth in FIG. 1.

Binding molecules, e.g., antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention, as known by those ofordinary skill in the art, can comprise a constant region which mediatesone or more effector functions. For example, binding of the C1 componentof complement to an antibody constant region may activate the complementsystem. Activation of complement is important in the opsonization andlysis of cell pathogens. The activation of complement also stimulatesthe inflammatory response and may also be involved in autoimmunehypersensitivity. Further, antibodies bind to receptors on various cellsvia the Fc region, with a Fc receptor binding site on the antibody Fcregion binding to a Fc receptor (FcR) on a cell. There are a number ofFc receptors which are specific for different classes of antibody,including IgG (gamma receptors), IgE (epsilon receptors), IgA (alphareceptors) and IgM (mu receptors). Binding of antibody to Fc receptorson cell surfaces triggers a number of important and diverse biologicalresponses including engulfment and destruction of antibody-coatedparticles, clearance of immune complexes, lysis of antibody-coatedtarget cells by killer cells (called antibody-dependent cell-mediatedcytotoxicity, or ADCC), release of inflammatory mediators, placentaltransfer and control of immunoglobulin production.

Accordingly, certain embodiments of the present invention include anantibody, or antigen-binding fragment, variant, or derivative thereof,in which at least a fraction of one or more of the constant regiondomains has been deleted or otherwise altered so as to provide desiredbiochemical characteristics such as reduced effector functions, theability to non-covalently dimerize, increased ability to localize at thesite of α-synuclein aggregation and deposition, reduced serum half-life,or increased serum half-life when compared with a whole, unalteredantibody of approximately the same immunogenicity. For example, certainantibodies for use in the diagnostic and treatment methods describedherein are domain deleted antibodies which comprise a polypeptide chainsimilar to an immunoglobulin heavy chain, but which lack at least aportion of one or more heavy chain domains. For instance, in certainantibodies, one entire domain of the constant region of the modifiedantibody will be deleted, for example, all or part of the CH2 domainwill be deleted. In other embodiments, certain antibodies for use in thediagnostic and treatment methods described herein have a constantregion, e.g., an IgG heavy chain constant region, which is altered toeliminate glycosylation, referred to elsewhere herein as aglycosylatedor “agly” antibodies. Such “agly” antibodies may be preparedenzymatically as well as by engineering the consensus glycosylationsite(s) in the constant region. While not being bound by theory, it isbelieved that “agly” antibodies may have an improved safety andstability profile in vivo. Methods of producing aglycosylatedantibodies, having desired effector function are found for example ininternational application W02005/018572, which is incorporated byreference in its entirety.

In certain antibodies, or antigen-binding fragments, variants, orderivatives thereof described herein, the Fc portion may be mutated todecrease effector function using techniques known in the art. Forexample, the deletion or inactivation (through point mutations or othermeans) of a constant region domain may reduce Fc receptor binding of thecirculating modified antibody thereby increasing α-synucleinlocalization. In other cases it may be that constant regionmodifications consistent with the instant invention moderate complementbinding and thus reduce the serum half life and nonspecific associationof a conjugated cytotoxin. Yet other modifications of the constantregion may be used to modify disulfide linkages or oligosaccharidemoieties that allow for enhanced localization due to increased antigenspecificity or antibody flexibility. The resulting physiologicalprofile, bioavailability and other biochemical effects of themodifications, such as α-synuclein localization, biodistribution andserum half-life, may easily be measured and quantified using well knowimmunological techniques without undue experimentation.

In certain antibodies, or antigen-binding fragments, variants, orderivatives thereof described herein, the Fc portion may be mutated orexchanged for alternative protein sequences to increase the cellularuptake of antibodies by way of example by enhancing receptor-mediatedendocytosis of antibodies via Fcy receptors, LRP, or Thy 1 receptors orby ‘SuperAntibody Technology’, which is said to enable antibodies to beshuttled into living cells without harming them (Expert Opin. Biol.Ther. (2005), 237-241). For example, the generation of fusion proteinsof the antibody binding region and the cognate protein ligands of cellsurface receptors or bi- or multi-specific antibodies with a specificsequences biding to α-synuclein as well as a cell surface receptor maybe engineered using techniques known in the art. In certain antibodies,or antigen-binding fragments, variants, or derivatives thereof describedherein, the Fc portion may be mutated or exchanged for alternativeprotein sequences or the antibody may be chemically modified to increaseits blood brain barrier penetration.

Modified forms of antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention can be made from whole precursor orparent antibodies using techniques known in the art. Exemplarytechniques are discussed in more detail herein. Antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention can be made or manufactured using techniques that are known inthe art. In certain embodiments, antibody molecules or fragments thereofare “recombinantly produced,” i.e., are produced using recombinant DNAtechnology. Exemplary techniques for making antibody molecules orfragments thereof are discussed in more detail elsewhere herein.

Antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention also include derivatives that are modified,e.g., by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody fromspecifically binding to its cognate epitope. For example, but not by wayof limitation, the antibody derivatives include antibodies that havebeen modified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

In particular preferred embodiments, antibodies, or antigen-bindingfragments, variants, or derivatives thereof of the invention will notelicit a deleterious immune response in the animal to be treated, e.g.,in a human. In certain embodiments, binding molecules, e.g., antibodies,or antigen-binding fragments thereof of the invention are derived from apatient, e.g., a human patient, and are subsequently used in the samespecies from which they are derived, e.g., human, alleviating orminimizing the occurrence of deleterious immune responses.

De-immunization can also be used to decrease the immunogenicity of anantibody. As used herein, the term “de-immunization” includes alterationof an antibody to modify T cell epitopes; see, e.g., internationalapplications WO98/52976 and WO00/34317. For example, V_(H) and V_(L)sequences from the starting antibody are analyzed and a human T cellepitope “map” from each V region showing the location of epitopes inrelation to complementarity determining regions (CDRs) and other keyresidues within the sequence. Individual T cell epitopes from the T cellepitope map are analyzed in order to identify alternative amino acidsubstitutions with a low risk of altering activity of the finalantibody. A range of alternative V_(H) and V_(L) sequences are designedcomprising combinations of amino acid substitutions and these sequencesare subsequently incorporated into a range of binding polypeptides,e.g., α-synuclein-specific antibodies or immunospecific fragmentsthereof for use in the diagnostic and treatment methods disclosedherein, which are then tested for function. Typically, between 12 and 24variant antibodies are generated and tested. Complete heavy and lightchain genes comprising modified V and human C regions are then clonedinto expression vectors and the subsequent plasmids introduced into celllines for the production of whole antibody. The antibodies are thencompared in appropriate biochemical and biological assays, and theoptimal variant is identified.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed.(1988); Hammerling et al., in: Monoclonal Antibodies and T-CellHybridomas Elsevier, N.Y., 563-681 (1981), said references incorporatedby reference in their entireties. The term “monoclonal antibody” as usedherein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced. Thus, the term“monoclonal antibody” is not limited to antibodies produced throughhybridoma technology. In certain embodiments, antibodies of the presentinvention are derived from human B cells which have been immortalizedvia transformation with Epstein-Barr virus, as described herein.

In the well known hybridoma process (Kohler et al., Nature 256 (1975),495) the relatively short-lived, or mortal, lymphocytes from a mammal,e.g., B cells derived from a human subject as described herein, arefused with an immortal tumor cell line (e.g.,. a myeloma cell line),thus, producing hybrid cells or “hybridomas” which are both immortal andcapable of producing the genetically coded antibody of the B cell. Theresulting hybrids are segregated into single genetic strains byselection, dilution, and re-growth with each individual straincomprising specific genes for the formation of a single antibody. Theyproduce antibodies, which are homogeneous against a desired antigen and,in reference to their pure genetic parentage, are termed “monoclonal”.

Hybridoma cells thus prepared are seeded and grown in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, parental myeloma cells. Those skilledin the art will appreciate that reagents, cell lines and media for theformation, selection and growth of hybridomas are commercially availablefrom a number of sources and standardized protocols are wellestablished. Generally, culture medium in which the hybridoma cells aregrowing is assayed for production of monoclonal antibodies against thedesired antigen. The binding specificity of the monoclonal antibodiesproduced by hybridoma cells is determined by in vitro assays such asimmunoprecipitation, radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA) as described herein. After hybridoma cellsare identified that produce antibodies of the desired specificity,affinity and/or activity, the clones may be subcloned by limitingdilution procedures and grown by standard methods; see, e.g., Goding,Monoclonal Antibodies: Principles and Practice, Academic Press, pp59-103 (1986). It will further be appreciated that the monoclonalantibodies secreted by the subclones may be separated from culturemedium, ascites fluid or serum by conventional purification proceduressuch as, for example, protein-A, hydroxylapatite chromatography, gelelectrophoresis, dialysis or affinity chromatography.

In another embodiment, lymphocytes can be selected by micromanipulationand the variable genes isolated. For example, peripheral bloodmononuclear cells can be isolated from an immunized or naturally immunemammal, e.g., a human, and cultured for about 7 days in vitro. Thecultures can be screened for specific IgGs that meet the screeningcriteria. Cells from positive wells can be isolated. IndividualIg-producing B cells can be isolated by FACS or by identifying them in acomplement-mediated hemolytic plaque assay. Ig-producing B cells can bemicromanipulated into a tube and the V_(H) and V_(L) genes can beamplified using, e.g., RT-PCR. The V_(H) and V_(L) genes can be clonedinto an antibody expression vector and transfected into cells (e.g.,eukaryotic or prokaryotic cells) for expression.

Alternatively, antibody-producing cell lines may be selected andcultured using techniques well known to the skilled artisan. Suchtechniques are described in a variety of laboratory manuals and primarypublications. In this respect, techniques suitable for use in theinvention as described below are described in Current Protocols inImmunology, Coligan et al., Eds., Green Publishing Associates andWiley-Interscience, John Wiley and Sons, New York (1991) which is hereinincorporated by reference in its entirety, including supplements.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)₂ fragments may be producedrecombinantly or by proteolytic cleavage of immunoglobulin molecules,using enzymes such as papain (to produce Fab fragments) or pepsin (toproduce F(ab′)₂ fragments). F(ab′)₂ fragments contain the variableregion, the light chain constant region and the CH1 domain of the heavychain. Such fragments are sufficient for use, for example, inimmunodiagnostic procedures involving coupling the immunospecificportions of immunoglobulins to detecting reagents such as radioisotopes.

Completely human antibodies, such as described herein, are particularlydesirable for therapeutic treatment of human patients. Human antibodiesof the present invention are isolated, e.g., from elderly subjects whobecause of their age may be suspected to be at risk of developing adisorder, e.g., Parkinson's disease, or a patient with the disorder butwith an unusually stable disease course. However, though it is prudentto expect that elderly healthy and symptom-free subjects, respectively,more regularly will have developed protective anti-α-synucleinantibodies than younger subjects, the latter may be used as well assource for obtaining a human antibody of the present invention. This isparticularly true for younger patients who are predisposed to develop afamilial form of a synucleinopathic disease but remain symptom-freesince their immune system and response functions more efficiently thanthat in older adults.

In one embodiment, an antibody of the invention comprises at least oneheavy or light chain CDR of an antibody molecule. In another embodiment,an antibody of the invention comprises at least two CDRs from one ormore antibody molecules. In another embodiment, an antibody of theinvention comprises at least three CDRs from one or more antibodymolecules. In another embodiment, an antibody of the invention comprisesat least four CDRs from one or more antibody molecules. In anotherembodiment, an antibody of the invention comprises at least five CDRsfrom one or more antibody molecules. In another embodiment, an antibodyof the invention comprises at least six CDRs from one or more antibodymolecules. Exemplary antibody molecules comprising at least one CDR thatcan be included in the subject antibodies are described herein.

Antibodies of the present invention can be produced by any method knownin the art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably by recombinant expression techniques asdescribed herein.

In one embodiment, an antibody, or antigen-binding fragment, variant, orderivative thereof of the invention comprises a synthetic constantregion wherein one or more domains are partially or entirely deleted(“domain-deleted antibodies”). In certain embodiments compatiblemodified antibodies will comprise domain deleted constructs or variantswherein the entire CH2 domain has been removed (ACH2 constructs). Forother embodiments a short connecting peptide may be substituted for thedeleted domain to provide flexibility and freedom of movement for thevariable region. Those skilled in the art will appreciate that suchconstructs are particularly preferred due to the regulatory propertiesof the CH2 domain on the catabolic rate of the antibody. Domain deletedconstructs can be derived using a vector encoding an IgGi human constantdomain, see, e.g., international applications WO02/060955 andWO02/096948A2. This vector is engineered to delete the CH2 domain andprovide a synthetic vector expressing a domain deleted IgGi constantregion.

In certain embodiments, antibodies, or antigen-binding fragments,variants, or derivatives thereof of the present invention areminibodies. Minibodies can be made using methods described in the art,see, e.g., U.S. Pat. No. 5,837,821 or international application WO94/09817.

In one embodiment, an antibody, or antigen-binding fragment, variant, orderivative thereof of the invention comprises an immunoglobulin heavychain having deletion or substitution of a few or even a single aminoacid as long as it permits association between the monomeric subunits.For example, the mutation of a single amino acid in selected areas ofthe CH2 domain may be enough to substantially reduce Fc binding andthereby increase α-synuclein localization. Similarly, it may bedesirable to simply delete that part of one or more constant regiondomains that control the effector function (e.g. complement binding) tobe modulated. Such partial deletions of the constant regions may improveselected characteristics of the antibody (serum half-life) while leavingother desirable functions associated with the subject constant regiondomain intact. Moreover, as alluded to above, the constant regions ofthe disclosed antibodies may be synthetic through the mutation orsubstitution of one or more amino acids that enhances the profile of theresulting construct. In this respect it may be possible to disrupt theactivity provided by a conserved binding site (e.g. Fc binding) whilesubstantially maintaining the configuration and immunogenic profile ofthe modified antibody. Yet other embodiments comprise the addition ofone or more amino acids to the constant region to enhance desirablecharacteristics such as effector function or provide for more cytotoxinor carbohydrate attachment. In such embodiments it may be desirable toinsert or replicate specific sequences derived from selected constantregion domains.

The present invention also provides antibodies that comprise, consistessentially of, or consist of, variants (including derivatives) ofantibody molecules (e.g., the V_(H) regions and/or V_(L) regions)described herein, which antibodies or fragments thereofimmunospecifically bind to α-synuclein. Standard techniques known tothose of skill in the art can be used to introduce mutations in thenucleotide sequence encoding an antibody, including, but not limited to,site-directed mutagenesis and PCR-mediated mutagenesis which result inamino acid substitutions. Preferably, the variants (includingderivatives) encode less than 50 amino acid substitutions, less than 40amino acid substitutions, less than 30 amino acid substitutions, lessthan 25 amino acid substitutions, less than 20 amino acid substitutions,less than 15 amino acid substitutions, less than 10 amino acidsubstitutions, less than 5 amino acid substitutions, less than 4 aminoacid substitutions, less than 3 amino acid substitutions, or less than 2amino acid substitutions relative to the reference V_(H) region,V_(H)-CDR1, V_(H)-CDR2, V_(H)-CDR3, V_(L)region, V_(L)-CDR1, V_(L)-CDR2,or V_(L)-CDR3. A “conservative amino acid substitution” is one in whichthe amino acid residue is replaced with an amino acid residue having aside chain with a similar charge. Families of amino acid residues havingside chains with similar charges have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Alternatively, mutations can beintroduced randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forbiological activity to identify mutants that retain activity (e.g., theability to bind α-synuclein).

For example, it is possible to introduce mutations only in frameworkregions or only in CDR regions of an antibody molecule. Introducedmutations may be silent or neutral missense mutations, e.g., have no, orlittle, effect on an antibody's ability to bind antigen, indeed somesuch mutations do not alter the amino acid sequence whatsoever. Thesetypes of mutations may be useful to optimize codon usage, or improve ahybridoma's antibody production. Codon-optimized coding regions encodingantibodies of the present invention are disclosed elsewhere herein.Alternatively, non-neutral missense mutations may alter an antibody'sability to bind antigen. The location of most silent and neutralmissense mutations is likely to be in the framework regions, while thelocation of most non-neutral missense mutations is likely to be in CDR,though this is not an absolute requirement. One of skill in the artwould be able to design and test mutant molecules with desiredproperties such as no alteration in antigen-binding activity oralteration in binding activity (e.g., improvements in antigen-bindingactivity or change in antibody specificity). Following mutagenesis, theencoded protein may routinely be expressed and the functional and/orbiological activity of the encoded protein, (e.g., ability toimmunospecifically bind at least one epitope of α-synuclein) can bedetermined using techniques described herein or by routinely modifyingtechniques known in the art.

III. Polynucleotides Encoding Antibodies

A polynucleotide encoding an antibody, or antigen-binding fragment,variant, or derivative thereof can be composed of any polyribonucleotideor polydeoxribonucleotide, which may be unmodified RNA or DNA ormodified RNA or DNA. For example, a polynucleotide encoding an antibody,or antigen-binding fragment, variant, or derivative thereof can becomposed of single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, a polynucleotide encoding an antibody, orantigen-binding fragment, variant, or derivative thereof can be composedof triple-stranded regions comprising RNA or DNA or both RNA and DNA. Apolynucleotide encoding an antibody, or antigen-binding fragment,variant, or derivative thereof may also contain one or more modifiedbases or DNA or RNA backbones modified for stability or for otherreasons. “Modified” bases include, for example, tritylated bases andunusual bases such as inosine. A variety of modifications can be made toDNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically,or metabolically modified forms.

An isolated polynucleotide encoding a non-natural variant of apolypeptide derived from an immunoglobulin (e.g., an immunoglobulinheavy chain portion or light chain portion) can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence of the immunoglobulin such that one or moreamino acid substitutions, additions or deletions are introduced into theencoded protein. Mutations may be introduced by standard techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis.Preferably, conservative amino acid substitutions are made at one ormore non-essential amino acid residues.

As is well known, RNA may be isolated from the original B cells,hybridoma cells or from other transformed cells by standard techniques,such as guanidinium isothiocyanate extraction and precipitation followedby centrifugation or chromatography. Where desirable, mRNA may beisolated from total RNA by standard techniques such as chromatography onoligo dT cellulose. Suitable techniques are familiar in the art. In oneembodiment, cDNAs that encode the light and the heavy chains of theantibody may be made, either simultaneously or separately, using reversetranscriptase and DNA polymerase in accordance with well known methods.PCR may be initiated by consensus constant region primers or by morespecific primers based on the published heavy and light chain DNA andamino acid sequences. As discussed above, PCR also may be used toisolate DNA clones encoding the antibody light and heavy chains. In thiscase the libraries may be screened by consensus primers or largerhomologous probes, such as human constant region probes.

DNA, typically plasmid DNA, may be isolated from the cells usingtechniques known in the art, restriction mapped and sequenced inaccordance with standard, well known techniques set forth in detail,e.g., in the foregoing references relating to recombinant DNAtechniques. Of course, the DNA may be synthetic according to the presentinvention at any point during the isolation process or subsequentanalysis.

In one embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin heavy chain variable region(V_(H)), where at least one of the CDRs of the heavy chain variableregion or at least two of the V_(H)-CDRs of the heavy chain variableregion are at least 80%, 85%, 90% or 95% identical to reference heavychain V_(H)-CDR1, V_(H)-CDR2, or V_(H)-CDR3 amino acid sequences fromthe antibodies disclosed herein. Alternatively, the V_(H)-CDR1,V_(H)-CDR2, or V_(H)-CDR3 regions of the V_(H) are at least 80%, 85%,90% or 95% identical to reference heavy chain V_(H)-CDR1, V_(H)-CDR2,and V_(H)-CDR3 amino acid sequences from the antibodies disclosedherein. Thus, according to this embodiment a heavy chain variable regionof the invention has V_(H)-CDR1, V_(H)-CDR2, or V_(H)-CDR3 polypeptidesequences related to the polypeptide sequences shown in FIG. 1.

In another embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin light chain variable region(V_(L)), where at least one of the V_(L)-CDRs of the light chainvariable region or at least two of the V_(L)-CDRs of the light chainvariable region are at least 80%, 85%, 90% or 95% identical to referencelight chain V_(L)-CDR1, V_(L)-CDR2, or V_(L)-CDR3 amino acid sequencesfrom the antibodies disclosed herein. Alternatively, the V_(L)-CDR1,V_(L)-CDR2, or V_(L)-CDR3 regions of the V_(L) are at least 80%, 85%,90% or 95% identical to reference light chain V_(L)-CDR1, V_(L)-CDR2,and V_(L)-CDR3 amino acid sequences from the antibodies disclosedherein. Thus, according to this embodiment a light chain variable regionof the invention has V_(L)-CDR1, V_(L)-CDR2, or V_(L)-CDR3 polypeptidesequences related to the polypeptide sequences shown in FIG. 1.

In another embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin heavy chain variable region(V_(H)) in which the V_(H)-CDR1, V_(H)-CDR2, and V_(H)-CDR3 regions havepolypeptide sequences which are identical to the V_(H)-CDR1, V_(H)-CDR2,and V_(H)-CDR3 groups shown in FIG. 1 and in the appended sequencelisting.

As known in the art, “sequence identity” between two polypeptides or twopolynucleotides is determined by comparing the amino acid or nucleicacid sequence of one polypeptide or polynucleotide to the sequence of asecond polypeptide or polynucleotide. When discussed herein, whether anyparticular polypeptide is at least about 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90% or 95% identical to another polypeptide can bedetermined using methods and computer programs/software known in the artsuch as, but not limited to, the BESTFIT program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711).BESTFIT uses the local homology algorithm of Smith and Waterman,Advances in Applied Mathematics 2 (1981), 482-489, to find the bestsegment of homology between two sequences. When using BESTFIT or anyother sequence alignment program to determine whether a particularsequence is, for example, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference polypeptide sequence and that gaps in homology of up to5% of the total number of amino acids in the reference sequence areallowed.

In a preferred embodiment of the present invention, the polynucleotidecomprises, consists essentially of, or consists of a nucleic acid havinga polynucleotide sequence of the V_(H) or V_(L) region of ananti-α-synuclein antibody as set forth in SEQ ID NOS: 2, 5, 8, 11, 14,17 or 20. In this respect, the person skilled in the art will readilyappreciate that the polynucleotides encoding at least the variabledomain of the light and/or heavy chain may encode the variable domain ofboth immunoglobulin chains or only one.

The present invention also includes fragments of the polynucleotides ofthe invention, as described elsewhere. Additionally polynucleotideswhich encode fusion polynucleotides, Fab fragments, and otherderivatives, as described herein, are also contemplated by theinvention.

The polynucleotides may be produced or manufactured by any method knownin the art. For example, if the nucleotide sequence of the antibody isknown, a polynucleotide encoding the antibody may be assembled fromchemically synthesized oligonucleotides, e.g., as described in Kutmeieret al., BioTechniques 17 (1994), 242, which, briefly, involves thesynthesis of overlapping oligonucleotides containing portions of thesequence encoding the antibody, annealing and ligating of thoseoligonucleotides, and then amplification of the ligated oligonucleotidesby PCR.

Alternatively, a polynucleotide encoding an antibody, or antigen-bindingfragment, variant, or derivative thereof may be generated from nucleicacid from a suitable source. If a clone containing a nucleic acidencoding a particular antibody is not available, but the sequence of theantibody molecule is known, a nucleic acid encoding the antibody may bechemically synthesized or obtained from a suitable source (e.g., anantibody cDNA library, or a cDNA library generated from, or nucleicacid, preferably polyA⁺ RNA, isolated from, any tissue or cellsexpressing the α-synuclein-specific antibody, such as hybridoma cellsselected to express an antibody) by PCR amplification using syntheticprimers hybridizable to the 3′ and 5′ ends of the sequence or by cloningusing an oligonucleotide probe specific for the particular gene sequenceto identify, e.g., a cDNA clone from a cDNA library that encodes theantibody. Amplified nucleic acids generated by PCR may then be clonedinto replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody, or antigen-binding fragment, variant, or derivativethereof is determined, its nucleotide sequence may be manipulated usingmethods well known in the art for the manipulation of nucleotidesequences, e.g., recombinant DNA techniques, site directed mutagenesis,PCR, etc. (see, for example, the techniques described in Sambrook etal., Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1990) and Ausubel et al., eds.,Current Protocols in Molecular Biology, John Wiley & Sons, NY (1998),which are both incorporated by reference herein in their entireties), togenerate antibodies having a different amino acid sequence, for exampleto create amino acid substitutions, deletions, and/or insertions.

IV. Expression of Antibody Polypeptides

Following manipulation of the isolated genetic material to provideantibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention, the polynucleotides encoding the antibodiesare typically inserted in an expression vector for introduction intohost cells that may be used to produce the desired quantity of antibody.Recombinant expression of an antibody, or fragment, derivative or analogthereof, e.g., a heavy or light chain of an antibody which binds to atarget molecule is described herein. Once a polynucleotide encoding anantibody molecule or a heavy or light chain of an antibody, or portionthereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g.,international applications WO 86/05807 and WO 89/01036; and U.S. Pat.No. 5,122,464) and the variable domain of the antibody may be clonedinto such a vector for expression of the entire heavy or light chain.

The term “vector” or “expression vector” is used herein to mean vectorsused in accordance with the present invention as a vehicle forintroducing into and expressing a desired gene in a host cell. As knownto those skilled in the art, such vectors may easily be selected fromthe group consisting of plasmids, phages, viruses and retroviruses. Ingeneral, vectors compatible with the instant invention will comprise aselection marker, appropriate restriction sites to facilitate cloning ofthe desired gene and the ability to enter and/or replicate in eukaryoticor prokaryotic cells. For the purposes of this invention, numerousexpression vector systems may be employed. For example, one class ofvector utilizes DNA elements which are derived from animal viruses suchas bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus,baculovirus, retroviruses (RSV, MMTV or MOMLV) or SV40 virus. Othersinvolve the use of polycistronic systems with internal ribosome bindingsites. Additionally, cells which have integrated the DNA into theirchromosomes may be selected by introducing one or more markers whichallow selection of transfected host cells. The marker may provide forprototrophy to an auxotrophic host, biocide resistance (e.g.,antibiotics) or resistance to heavy metals such as copper. Theselectable marker gene can either be directly linked to the DNAsequences to be expressed, or introduced into the same cell byco-transformation. Additional elements may also be needed for optimalsynthesis of mRNA. These elements may include signal sequences, splicesignals, as well as transcriptional promoters, enhancers, andtermination signals.

In particularly preferred embodiments the cloned variable region genesare inserted into an expression vector along with the heavy and lightchain constant region genes (preferably human) as discussed above. Inone embodiment, this is effected using a proprietary expression vectorof Biogen IDEC, Inc., referred to as NEOSPLA, disclosed in U.S. Pat No.6,159,730. This vector contains the cytomegalovirus promoter/enhancer,the mouse beta globin major promoter, the SV40 origin of replication,the bovine growth hormone polyadenylation sequence, neomycinphosphotransferase exon 1 and exon 2, the dihydrofolate reductase geneand leader sequence. This vector has been found to result in very highlevel expression of antibodies upon incorporation of variable andconstant region genes, transfection in CHO cells, followed by selectionin G418 containing medium and methotrexate amplification. Of course, anyexpression vector which is capable of eliciting expression in eukaryoticcells may be used in the present invention. Examples of suitable vectorsinclude, but are not limited to plasmids pcDNA3, pHCMV/Zeo, pCR3.1,pEF1/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV, pUB6N5-His,pVAX1, and pZeoSV2 (available from Invitrogen, San Diego, Calif.), andplasmid pCI (available from Promega, Madison, Wis.). In general,screening large numbers of transformed cells for those which expresssuitably high levels if immunoglobulin heavy and light chains is routineexperimentation which can be carried out, for example, by roboticsystems. Vector systems are also taught in U.S. Pat. Nos. 5,736,137 and5,658,570, each of which is incorporated by reference in its entiretyherein. This system provides for high expression levels, e.g., >30pg/cell/day. Other exemplary vector systems are disclosed e.g., in U.S.Pat. No. 6,413,777.

In other preferred embodiments the antibodies, or antigen-bindingfragments, variants, or derivatives thereof of the invention may beexpressed using polycistronic constructs such as those disclosed in USpatent application publication no. 2003-0157641 A1 and incorporatedherein in its entirety. In these expression systems, multiple geneproducts of interest such as heavy and light chains of antibodies may beproduced from a single polycistronic construct. These systemsadvantageously use an internal ribosome entry site (IRES) to providerelatively high levels of antibodies. Compatible IRES sequences aredisclosed in U.S. Pat. No. 6,193,980 which is also incorporated herein.Those skilled in the art will appreciate that such expression systemsmay be used to effectively produce the full range of antibodiesdisclosed in the instant application.

More generally, once the vector or DNA sequence encoding a monomericsubunit of the antibody has been prepared, the expression vector may beintroduced into an appropriate host cell. Introduction of the plasmidinto the host cell can be accomplished by various techniques well knownto those of skill in the art. These include, but are not limited to,transfection including lipotransfection using, e.g., Fugene orlipofectamine, protoplast fusion, calcium phosphate precipitation, cellfusion with enveloped DNA, microinjection, and infection with intactvirus. Typically, plasmid introduction into the host is via standardcalcium phosphate co-precipitation method. The host cells harboring theexpression construct are grown under conditions appropriate to theproduction of the light chains and heavy chains, and assayed for heavyand/or light chain protein synthesis. Exemplary assay techniques includeenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), orfluorescence-activated cell sorter analysis (FACS), immunohistochemistryand the like.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody for use in the methods describedherein. Thus, the invention includes host cells containing apolynucleotide encoding an antibody of the invention, or a heavy orlight chain thereof, operably linked to a heterologous promoter. Inpreferred embodiments for the expression of double-chained antibodies,vectors encoding both the heavy and light chains may be co-expressed inthe host cell for expression of the entire immunoglobulin molecule, asdetailed below.

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes both heavy and light chainpolypeptides. In such situations, the light chain is advantageouslyplaced before the heavy chain to avoid an excess of toxic free heavychain; see Proudfoot, Nature 322 (1986), 52; Kohler, Proc. Natl. Acad.Sci. USA 77 (1980), 2197. The coding sequences for the heavy and lightchains may comprise cDNA or genomic DNA.

As used herein, “host cells” refers to cells which harbor vectorsconstructed using recombinant DNA techniques and encoding at least oneheterologous gene. In descriptions of processes for isolation ofantibodies from recombinant hosts, the terms “cell” and “cell culture”are used interchangeably to denote the source of antibody unless it isclearly specified otherwise. In other words, recovery of polypeptidefrom the “cells” may mean either from spun down whole cells, or from thecell culture containing both the medium and the suspended cells.

A variety of host-expression vector systems may be utilized to expressantibody molecules for use in the methods described herein. Suchhost-expression systems represent vehicles by which the coding sequencesof interest may be produced and subsequently purified, but alsorepresent cells which may, when transformed or transfected with theappropriate nucleotide coding sequences, express an antibody molecule ofthe invention in situ. These include but are not limited tomicroorganisms such as bacteria (e.g., E. coli, B. subtilis) transformedwith recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expressionvectors containing antibody coding sequences; yeast (e.g.,Saccharomyces, Pichia) transformed with recombinant yeast expressionvectors containing antibody coding sequences; insect cell systemsinfected with recombinant virus expression vectors (e.g., baculovirus)containing antibody coding sequences; plant cell systems infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing antibody codingsequences; or mammalian cell systems (e.g., COS, CHO, NSO, BLK, 293, 3T3cells) harboring recombinant expression constructs containing promotersderived from the genome of mammalian cells (e.g., metallothioneinpromoter) or from mammalian viruses (e.g., the adenovirus late promoter;the vaccinia virus 7.5K promoter). Preferably, bacterial cells such asEscherichia coli, and more preferably, eukaryotic cells, especially forthe expression of whole recombinant antibody molecule, are used for theexpression of a recombinant antibody molecule. For example, mammaliancells such as Chinese Hamster Ovary (CHO) cells, in conjunction with avector such as the major intermediate early gene promoter element fromhuman cytomegalovirus is an effective expression system for antibodies;see, e.g., Foecking et al., Gene 45 (1986), 101; Cockett et al.,Bio/Technology 8 (1990), 2.

The host cell line used for protein expression is often of mammalianorigin; those skilled in the art are credited with ability topreferentially determine particular host cell lines which are bestsuited for the desired gene product to be expressed therein. Exemplaryhost cell lines include, but are not limited to, CHO (Chinese HamsterOvary), DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFR minus), HELA(human cervical carcinoma), CVI (monkey kidney line), COS (a derivativeof CVI with SV40 T antigen), VERY, BHK (baby hamster kidney), MDCK,WI38, R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast),HAK (hamster kidney line), SP2/O (mouse myeloma), P3x63-Ag3.653 (mousemyeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocyte)and 293 (human kidney). CHO and 293 cells are particularly preferred.Host cell lines are typically available from commercial services, theAmerican Tissue Culture Collection or from published literature.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which stably express theantibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11(1977), 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska& Szybalski, Proc. Natl. Acad. Sci. USA 48 (1992), 202), and adeninephosphoribosyltransferase (Lowy et al., Cell 22 (1980), 817) genes canbe employed in tk-, hgprt- or aprt-cells, respectively. Also,anti-metabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77 (1980), 357; O'Hare et al., Proc. Natl.Acad. Sci. USA 78 (1981), 1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78(1981), 2072); neo, which confers resistance to the aminoglycoside G-418Goldspiel et al., Clinical Pharmacy 12 (1993), 488-505; Wu and Wu,Biotherapy 3 (1991), 87-95; Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32(1993), 573-596; Mulligan, Science 260 (1993), 926-932; and Morgan andAnderson, Ann. Rev. Biochem. 62 (1993), 191-217; TIB TECH 11 (1993),155-215; and hygro, which confers resistance to hygromycin (Santerre etal., Gene 30 (1984), 147. Methods commonly known in the art ofrecombinant DNA technology which can be used are described in Ausubel etal. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons,NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual,Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al.(eds), Current Protocols in Human Genetics, John Wiley & Sons, NY(1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which areincorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification, for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Academic Press, New York, Vol. 3.(1987). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase; see Crouse et al., Mol. Cell. Biol. 3(1983), 257.

In vitro production allows scale-up to give large amounts of the desiredpolypeptides. Techniques for mammalian cell cultivation under tissueculture conditions are known in the art and include homogeneoussuspension culture, e.g. in an airlift reactor or in a continuousstirrer reactor, or immobilized or entrapped cell culture, e.g. inhollow fibers, microcapsules, on agarose microbeads or ceramiccartridges. If necessary and/or desired, the solutions of polypeptidescan be purified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose or (immuno-)affinity chromatography, e.g., afterpreferential biosynthesis of a synthetic hinge region polypeptide orprior to or subsequent to the HIC chromatography step described herein.

Genes encoding antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention can also be expressed innon-mammalian cells such as bacteria or insect or yeast or plant cells.Bacteria which readily take up nucleic acids include members of theenterobacteriaceae, such as strains of Escherichia coli or Salmonella;Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, andHaemophilus influenzae. It will further be appreciated that, whenexpressed in bacteria, the heterologous polypeptides typically becomepart of inclusion bodies. The heterologous polypeptides must beisolated, purified and then assembled into functional molecules. Wheretetravalent forms of antibodies are desired, the subunits will thenself-assemble into tetravalent antibodies; see, e.g., internationalapplication WO02/096948.

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2 (1983),1791), in which the antibody coding sequence may be ligated individuallyinto the vector in frame with the lacZ coding region so that a fusionprotein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13(1985), 3101-3109; Van Heeke & Schuster, J. Biol. Chem. 24 (1989),5503-5509); and the like. pGEX vectors may also be used to expressforeign polypeptides as fusion proteins with glutathione S-transferase(GST). In general, such fusion proteins are soluble and can easily bepurified from lysed cells by adsorption and binding to a matrix ofglutathione-agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target gene product can bereleased from the GST moiety.

In addition to prokaryotes, eukaryotic microbes may also be used.Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among eukaryotic microorganisms although a number of other strainsare commonly available, e.g., Pichia pastoris. For expression inSaccharomyces, the plasmid YRp7, for example, (Stinchcomb et al., Nature282 (1979), 39; Kingsman et al., Gene 7 (1979), 141; Tschemper et al.,Gene 10 (1980), 157) is commonly used. This plasmid already contains theTRP1 gene which provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example ATCC No. 44076 orPEP4-1 (Jones, Genetics 85 (1977), 12). The presence of the trpl lesionas a characteristic of the yeast host cell genome then provides aneffective environment for detecting transformation by growth in theabsence of tryptophan.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is typically used as a vector to express foreign genes. Thevirus grows in Spodoptera frugiperda cells. The antibody coding sequencemay be cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter).

Once an antibody molecule of the invention has been recombinantlyexpressed, the whole antibodies, their dimers, individual light andheavy chains, or other immunoglobulin forms of the present invention,can be purified according to standard procedures of the art, includingfor example, by chromatography (e.g., ion exchange, affinity,particularly by affinity for the specific antigen after Protein A, andsizing column chromatography), centrifugation, differential solubility,e.g. ammonium sulfate precipitation, or by any other standard techniquefor the purification of proteins; see, e.g., Scopes, “ProteinPurification”, Springer Verlag, N.Y. (1982). Alternatively, a preferredmethod for increasing the affinity of antibodies of the invention isdisclosed in US patent publication 2002-0123057 A1.

V. Fusion Proteins and Conjugates

In certain embodiments, the antibody polypeptide comprises an amino acidsequence or one or more moieties not normally associated with anantibody. Exemplary modifications are described in more detail below.For example, a single-chain fv antibody fragment of the invention maycomprise a flexible linker sequence, or may be modified to add afunctional moiety (e.g., PEG, a drug, a toxin, or a label such as afluorescent, radioactive, enzyme, nuclear magnetic, heavy metal and thelike)

An antibody polypeptide of the invention may comprise, consistessentially of, or consist of a fusion protein. Fusion proteins arechimeric molecules which comprise, for example, an immunoglobulinα-synuclein-binding domain with at least one target binding site, and atleast one heterologous portion, i.e., a portion with which it is notnaturally linked in nature. The amino acid sequences may normally existin separate proteins that are brought together in the fusion polypeptideor they may normally exist in the same protein but are placed in a newarrangement in the fusion polypeptide. Fusion proteins may be created,for example, by chemical synthesis, or by creating and translating apolynucleotide in which the peptide regions are encoded in the desiredrelationship.

The term “heterologous” as applied to a polynucleotide or a polypeptide,means that the polynucleotide or polypeptide is derived from a distinctentity from that of the rest of the entity to which it is beingcompared. For instance, as used herein, a “heterologous polypeptide” tobe fused to an antibody, or an antigen-binding fragment, variant, oranalog thereof is derived from a non-immunoglobulin polypeptide of thesame species, or an immunoglobulin or non-immunoglobulin polypeptide ofa different species. As discussed in more detail elsewhere herein,antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalent and non-covalent conjugations) topolypeptides or other compositions. For example, antibodies may berecombinantly fused or conjugated to molecules useful as labels indetection assays and effector molecules such as heterologouspolypeptides, drugs, radionuclides, or toxins; see, e.g., internationalapplications WO92/08495; WO91/14438; WO89/12624; U.S. Pat. No.5,314,995; and European patent application EP 0 396 387.

Antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention can be composed of amino acids joined to eachother by peptide bonds or modified peptide bonds, i.e., peptideisosteres, and may contain amino acids other than the 20 gene-encodedamino acids. Antibodies may be modified by natural processes, such asposttranslational processing, or by chemical modification techniqueswhich are well known in the art. Such modifications are well describedin basic texts and in more detailed monographs, as well as in avoluminous research literature. Modifications can occur anywhere in theantibody, including the peptide backbone, the amino acid side-chains andthe amino or carboxyl termini, or on moieties such as carbohydrates. Itwill be appreciated that the same type of modification may be present inthe same or varying degrees at several sites in a given antibody. Also,a given antibody may contain many types of modifications. Antibodies maybe branched, for example, as a result of ubiquitination, and they may becyclic, with or without branching. Cyclic, branched, and branched cyclicantibodies may result from posttranslation natural processes or may bemade by synthetic methods. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, transfer-RNA mediated additionof amino acids to proteins such as arginylation, and ubiquitination;see, e.g., Proteins-Structure And Molecular Properties, T. E. Creighton,W. H. Freeman and Company, New York 2nd Ed., (1993); PosttranslationalCovalent Modification Of Proteins, B. C. Johnson, Ed., Academic Press,New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol. 182 (1990),626-646; Rattan et al., Ann. NY Acad. Sci. 663 (1992), 48-62).

The present invention also provides for fusion proteins comprising anantibody, or antigen-binding fragment, variant, or derivative thereof,and a heterologous polypeptide. In one embodiment, a fusion protein ofthe invention comprises, consists essentially of, or consists of, apolypeptide having the amino acid sequence of any one or more of theV_(H) regions of an antibody of the invention or the amino acid sequenceof any one or more of the V_(L) regions of an antibody of the inventionor fragments or variants thereof, and a heterologous polypeptidesequence. In another embodiment, a fusion protein for use in thediagnostic and treatment methods disclosed herein comprises, consistsessentially of, or consists of a polypeptide having the amino acidsequence of any one, two, three of the V_(H)-CDRs of an antibody, orfragments, variants, or derivatives thereof, or the amino acid sequenceof any one, two, three of the V_(L)-CDRs of an antibody, or fragments,variants, or derivatives thereof, and a heterologous polypeptidesequence. In one embodiment, the fusion protein comprises a polypeptidehaving the amino acid sequence of a V_(H)-CDR3 of an antibody of thepresent invention, or fragment, derivative, or variant thereof, and aheterologous polypeptide sequence, which fusion protein specificallybinds to α-synuclein. In another embodiment, a fusion protein comprisesa polypeptide having the amino acid sequence of at least one V_(H)region of an antibody of the invention and the amino acid sequence of atleast one V_(L) region of an antibody of the invention or fragments,derivatives or variants thereof, and a heterologous polypeptidesequence. Preferably, the V_(H) and V_(L) regions of the fusion proteincorrespond to a single source antibody (or scFv or Fab fragment) whichspecifically binds α-synuclein. In yet another embodiment, a fusionprotein for use in the diagnostic and treatment methods disclosed hereincomprises a polypeptide having the amino acid sequence of any one, two,three or more of the V_(H) CDRs of an antibody and the amino acidsequence of any one, two, three or more of the V_(L) CDRs of anantibody, or fragments or variants thereof, and a heterologouspolypeptide sequence. Preferably, two, three, four, five, six, or moreof the V_(H)-CDR(s) or V_(L)-CDR(s) correspond to single source antibody(or scFv or Fab fragment) of the invention. Nucleic acid moleculesencoding these fusion proteins are also encompassed by the invention.

Exemplary fusion proteins reported in the literature include fusions ofthe T cell receptor (Gascoigne et al., Proc. Natl. Acad. Sci. USA 84(1987), 2936-2940; CD4 (Capon et al., Nature 337 (1989), 525-531;Traunecker et al., Nature 339 (1989), 68-70; Zettmeissl et al., DNA CellBiol. USA 9 (1990), 347-353; and Byrn et al., Nature 344 (1990),667-670); L-selectin (homing receptor) (Watson et al., J. Cell. Biol.110 (1990), 2221-2229; and Watson et al., Nature 349 (1991), 164-167);CD44 (Aruffo et al., Cell 61 (1990), 1303-1313); CD28 and B7 (Linsley etal., J. Exp. Med. 173 (1991),721-730); CTLA-4 (Lisley et al., J. Exp.Med. 174 (1991), 561-569); CD22 (Stamenkovic et al., Cell 66 (1991),1133-1144); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA88 (1991), 10535-10539; Lesslauer et al., Eur. J. Immunol. 27 (1991),2883-2886; and Peppel et al., J. Exp. Med. 174 (1991), 1483-1489 (1991);and IgE receptor a (Ridgway and Gorman, J. Cell. Biol. 115 (1991),Abstract No. 1448).

As discussed elsewhere herein, antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention may be fused toheterologous polypeptides to increase the in vivo half life of thepolypeptides or for use in immunoassays using methods known in the art.For example, in one embodiment, PEG can be conjugated to the antibodiesof the invention to increase their half-life in vivo; see, e.g., Leonget al., Cytokine 16 (2001), 106-119; Adv. in Drug Deliv. Rev. 54 (2002),531; or Weir et al., Biochem. Soc. Transactions 30 (2002), 512.

Moreover, antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention can be fused to marker sequences,such as a peptide to facilitate their purification or detection. Inpreferred embodiments, the marker amino acid sequence is ahexa-histidine peptide (HIS), such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif. , 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86 (1989), 821-824, for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37 (1984), 767)and the “flag” tag.

Fusion proteins can be prepared using methods that are well known in theart; see for example U.S. Pat. Nos. 5,116,964 and 5,225,538. The precisesite at which the fusion is made may be selected empirically to optimizethe secretion or binding characteristics of the fusion protein. DNAencoding the fusion protein is then transfected into a host cell forexpression.

Antibodies of the present invention may be used in non-conjugated formor may be conjugated to at least one of a variety of molecules, e.g., toimprove the therapeutic properties of the molecule, to facilitate targetdetection, or for imaging or therapy of the patient. Antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention can be labeled or conjugated either before or afterpurification, when purification is performed. In particular, antibodies,or antigen-binding fragments, variants, or derivatives thereof of theinvention may be conjugated to therapeutic agents, prodrugs, peptides,proteins, enzymes, viruses, lipids, biological response modifiers,pharmaceutical agents, or PEG.

Conjugates that are immunotoxins including conventional antibodies havebeen widely described in the art. The toxins may be coupled to theantibodies by conventional coupling techniques or immunotoxinscontaining protein toxin portions can be produced as fusion proteins.The antibodies of the present invention can be used in a correspondingway to obtain such immunotoxins. Illustrative of such immunotoxins arethose described by Byers, Seminars Cell. Biol. 2 (1991), 59-70 and byFanger, Immunol. Today 12 (1991), 51-54.

Those skilled in the art will appreciate that conjugates may also beassembled using a variety of techniques depending on the selected agentto be conjugated. For example, conjugates with biotin are prepared e.g.by reacting an α-synuclein binding polypeptide with an activated esterof biotin such as the biotin N-hydroxysuccinimide ester. Similarly,conjugates with a fluorescent marker may be prepared in the presence ofa coupling agent, e.g. those listed herein, or by reaction with anisothiocyanate, preferably fluorescein-isothiocyanate. Conjugates of theantibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention are prepared in an analogous manner.

The present invention further encompasses antibodies, or antigen-bindingfragments, variants, or derivatives thereof of the invention conjugatedto a diagnostic or therapeutic agent. The antibodies can be useddiagnostically to, for example, demonstrate presence of a neurologicaldisease, to indicate the risk of getting a neurological disease, tomonitor the development or progression of a neurological disease, i.e.synucleinopathic disease as part of a clinical testing procedure to,e.g., determine the efficacy of a given treatment and/or preventionregimen. Detection can be facilitated by coupling the antibody, orantigen-binding fragment, variant, or derivative thereof to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, radioactive materials, positron emittingmetals using various positron emission tomographies, and nonradioactiveparamagnetic metal ions; see, e.g., U.S. Pat. No. 4,741,900 for metalions which can be conjugated to antibodies for use as diagnosticsaccording to the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ¹¹¹In or ⁹⁹Tc.

An antibody, or antigen-binding fragment, variant, or derivative thereofalso can be detectably labeled by coupling it to a chemiluminescentcompound. The presence of the chemiluminescent-tagged antibody is thendetermined by detecting the presence of luminescence that arises duringthe course of a chemical reaction. Examples of particularly usefulchemiluminescent labeling compounds are luminol, isoluminol, theromaticacridinium ester, imidazole, acridinium salt and oxalate ester.

One of the ways in which an antibody, or antigen-binding fragment,variant, or derivative thereof can be detectably labeled is by linkingthe same to an enzyme and using the linked product in an enzymeimmunoassay (EIA) (Voller, A., “The Enzyme Linked Immunosorbent Assay(ELISA)” Microbiological Associates Quarterly Publication, Walkersville,Md., Diagnostic Horizons 2 (1978), 1-7); Voller et al., J. Clin. Pathol.31 (1978), 507-520; Butler, Meth. Enzymol. 73 (1981), 482-523; Maggio,E. (ed.), Enzyme Immunoassay, CRC Press, Boca Raton, Fla., (1980);Ishikawa, E. et al., (eds.), Enzyme Immunoassay, Kgaku Shoin, Tokyo(1981). The enzyme, which is bound to the antibody will react with anappropriate substrate, preferably a chromogenic substrate, in such amanner as to produce a chemical moiety which can be detected, forexample, by spectrophotometric, fluorimetric or by visual means. Enzymeswhich can be used to detectably label the antibody include, but are notlimited to, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. Additionally, the detection can be accomplished bycolorimetric methods which employ a chromogenic substrate for theenzyme. Detection may also be accomplished by visual comparison of theextent of enzymatic reaction of a substrate in comparison with similarlyprepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibody, orantigen-binding fragment, variant, or derivative thereof, it is possibleto detect the antibody through the use of a radioimmunoassay (RIA) (see,for example, Weintraub, B., Principles of Radioimmunoassays, SeventhTraining Course on Radioligand Assay Techniques, The Endocrine Society,(March, 1986)), which is incorporated by reference herein). Theradioactive isotope can be detected by means including, but not limitedto, a gamma counter, a scintillation counter, or autoradiography.

An antibody, or antigen-binding fragment, variant, or derivative thereofcan also be detectably labeled using fluorescence emitting metals suchas ¹⁵²Eu, or others of the lanthanide series. These metals can beattached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

Techniques for conjugating various moieties to an antibody, orantigen-binding fragment, variant, or derivative thereof are well known,see, e.g., Amon et al., “Monoclonal Antibodies For Immunotargeting OfDrugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy,Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstromet al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2ndEd.), Robinson et al. (eds.), Marcel Dekker, Inc., pp. 623-53 (1987);Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: AReview”, in Monoclonal Antibodies '84: Biological And ClinicalApplications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis,Results, And Future Prospective Of The Therapeutic Use Of RadiolabeledAntibody In Cancer Therapy”, in Monoclonal Antibodies For CancerDetection And Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16(1985), and Thorpe et al., “The Preparation And Cytotoxic Properties OfAntibody-Toxin Conjugates”, Immunol. Rev. 62 (1982), 119-158.

As mentioned, in certain embodiments, a moiety that enhances thestability or efficacy of a binding molecule, e.g., a bindingpolypeptide, e.g., an antibody or immunospecific fragment thereof can beconjugated. For example, in one embodiment, PEG can be conjugated to thebinding molecules of the invention to increase their half-life in vivo.Leong et al., Cytokine 16 (2001), 106; Adv. in Drug Deliv. Rev. 54(2002), 531; or Weir et al., Biochem. Soc. Transactions 30 (2002), 512.

VI. Compositions and Methods of Use

The present invention relates to compositions comprising theaforementioned α-synuclein binding molecule, e.g., antibody orantigen-binding fragment thereof of the present invention or derivativeor variant thereof, or the polynucleotide, vector or cell of theinvention. The composition of the present invention may further comprisea pharmaceutically acceptable carrier. Furthermore, the pharmaceuticalcomposition of the present invention may comprise further agents such asinterleukins or interferons depending on the intended use of thepharmaceutical composition. For example, for use in the treatment ofParkinson's disease the additional agent may be selected from the groupconsisting of small organic molecules, anti-α-synuclein antibodies, andcombinations thereof. Hence, in a particular preferred embodiment thepresent invention relates to the use of the α-synuclein bindingmolecule, e.g., antibody or antigen-binding fragment thereof of thepresent invention or of a binding molecule having substantially the samebinding specificities of any one thereof, the polynucleotide, the vectoror the cell of the present invention for the preparation of apharmaceutical or diagnostic composition for prophylactic andtherapeutic treatment of a synucleinopathic disease, monitoring theprogression of a synucleinopathic disease or a response to asynucleinopathic disease treatment in a subject or for determining asubject's risk for developing a synucleinopathic disease.

Hence, in one embodiment the present invention relates to a method oftreating a neurological disorder characterized by abnormal accumulationand/or deposition of α-synuclein in the brain and the central nervoussystem, respectively, which method comprises administering to a subjectin need thereof a therapeutically effective amount of any one of theafore-described α-synuclein binding molecules, antibodies,polynucleotides, vectors or cells of the instant invention. The term“neurological disorder” includes but is not limited to synucleinopathicdiseases such as Parkinson's disease (PD), Parkinson's disease dementia(PDD), dementia with Lewy bodies (DLB), the Lewy body variant ofAlzheimer's disease (LBVAD), multiple systems atrophy (MSA), pureautonomic failure (PAF), neurodegeneration with brain iron accumulationtype-1 (NBIA-I), Alzheimer's disease, Pick disease, juvenile-onsetgeneralized neuroaxonal dystrophy (Hallervorden-Spatz disease),amyotrophic lateral sclerosis, traumatic brain injury, and Down syndromeas well as other movement disorders and disease of the central nervoussystem (CNS) in general. Unless stated otherwise, the termsneurodegenerative, neurological or neuropsychiatric are usedinterchangeably herein.

A particular advantage of the therapeutic approach of the presentinvention lies in the fact that the antibodies of the present inventionare derived from B cells or B memory cells from elderly subjects with nosigns of Parkinsonism and thus are, with a certain probability, capableof preventing a clinically manifest synucleinopathic disease, or ofdiminishing the risk of the occurrence of the clinically manifestdisease, or of delaying the onset or progression of the clinicallymanifest disease. Typically, the antibodies of the present inventionalso have already successfully gone through somatic maturation, i.e. theoptimization with respect to selectivity and effectiveness in the highaffinity binding to the target α-synuclein molecule by means of somaticvariation of the variable regions of the antibody.

The knowledge that such cells in vivo, e.g. in a human, have not beenactivated by means of related or other physiological proteins or cellstructures in the sense of an autoimmunological or allergic reaction isalso of great medical importance since this signifies a considerablyincreased chance of successfully living through the clinical testphases. So to speak, efficiency, acceptability and tolerability havealready been demonstrated before the preclinical and clinicaldevelopment of the prophylactic or therapeutic antibody in at least onehuman subject. It can thus be expected that the human anti-α-synucleinantibodies of the present invention, both its target structure-specificefficiency as therapeutic agent and its decreased probability of sideeffects significantly increase its clinical probability of success.

The present invention also provides a pharmaceutical and diagnostic,respectively, pack or kit comprising one or more containers filled withone or more of the above described ingredients, e.g. anti-α-synucleinantibody, binding fragment, derivative or variant thereof,polynucleotide, vector or cell of the present invention. Associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. Inaddition or alternatively the kit comprises reagents and/or instructionsfor use in appropriate diagnostic assays. The composition, e.g. kit ofthe present invention is of course particularly suitable for the riskassessment, diagnosis, prevention and treatment of a disorder which isaccompanied with the presence of α-synuclein, and in particularapplicable for the treatment of Parkinson's disease (PD), Parkinson'sdisease dementia (PDD), dementia with Lewy bodies (DLB) and Lewy bodyvariant of Alzheimer's disease (LBVAD).

The pharmaceutical compositions of the present invention can beformulated according to methods well known in the art; see for exampleRemington: The Science and Practice of Pharmacy (2000) by the Universityof Sciences in Philadelphia, ISBN 0-683-306472. Examples of suitablepharmaceutical carriers are well known in the art and include phosphatebuffered saline solutions, water, emulsions, such as oil/wateremulsions, various types of wetting agents, sterile solutions etc.Compositions comprising such carriers can be formulated by well knownconventional methods. These pharmaceutical compositions can beadministered to the subject at a suitable dose. Administration of thesuitable compositions may be effected by different ways, e.g., byintravenous, intraperitoneal, subcutaneous, intramuscular, intranasal,topical or intradermal administration or spinal or brain delivery.Aerosol formulations such as nasal spray formulations include purifiedaqueous or other solutions of the active agent with preservative agentsand isotonic agents. Such formulations are preferably adjusted to a pHand isotonic state compatible with the nasal mucous membranes.Formulations for rectal or vaginal ad-ministration may be presented as asuppository with a suitable carrier.

Furthermore, whereas the present invention includes the now standard(though fortunately infrequent) procedure of drilling a small hole inthe skull to administer a drug of the present invention, in a preferredaspect, the binding molecule, especially antibody or antibody based drugof the present invention can cross the blood-brain barrier, which allowsfor intravenous or oral administration.

The dosage regimen will be determined by the attending physician andclinical factors. As is well known in the medical arts, dosages for anyone patient depends upon many factors, including the patient's size,body surface area, age, the particular compound to be administered, sex,time and route of administration, general health, and other drugs beingadministered concurrently. A typical dose can be, for example, in therange of 0.001 to 1000 μg (or of nucleic acid for expression or forinhibition of expression in this range); however, doses below or abovethis exemplary range are envisioned, especially considering theaforementioned factors. Generally, the dosage can range, e.g., fromabout 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg (e.g., 0.02mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, etc.), ofthe host body weight. For example dosages can be 1 mg/kg body weight or10 mg/kg body weight or within the range of 1-10 mg/kg, preferably atleast 1 mg/kg. Doses intermediate in the above ranges are also intendedto be within the scope of the invention. Subjects can be administeredsuch doses daily, on alternative days, weekly or according to any otherschedule determined by empirical analysis. An exemplary treatmententails administration in multiple dosages over a prolonged period, forexample, of at least six months. Additional exemplary treatment regimesentail administration once per every two weeks or once a month or onceevery 3 to 6 months. Exemplary dosage schedules include 1-10 mg/kg or 15mg/kg on consecutive days, 30 mg/kg on alternate days or 60 mg/kgweekly. In some methods, two or more monoclonal antibodies withdifferent binding specificities are administered simultaneously, inwhich case the dosage of each antibody administered falls within theranges indicated. Progress can be monitored by periodic assessment.Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Furthermore, the pharmaceutical composition of the invention maycomprise further agents such as dopamine or psychopharmacologic drugs,depending on the intended use of the pharmaceutical composition.

Furthermore, in a preferred embodiment of the present invention thepharmaceutical composition may be formulated as a vaccine, for example,if the pharmaceutical composition of the invention comprises ananti-α-synuclein antibody or binding fragment, derivative or variantthereof for passive immunization. As mentioned in the backgroundsection, oligomeric species of α-synuclein have been reportedextracellularly in plasma and CSF (El-Agnaf et al., FASEB J. 20 (2006),419-425) and passive immunization studies in mouse models of Parkinson'sdisease show that extracellular mouse monoclonal antibodies againstα-synuclein can reduce accumulation of intracellular α-synucleinaggregates (Masliah et al., Neuron, 46 (2005), 857-868). Accordingly itis prudent to expect that the human anti-α-synuclein antibodies andequivalent α-synuclein binding molecules of the present invention areparticularly useful as a vaccine for the prevention or amelioration ofsynucleinopathic diseases such as PD, DLB and LBVAD.

In one embodiment, it may be beneficial to use recombinant Fab (rFab)and single chain fragments (scFvs) of the antibody of the presentinvention, which might more readily penetrate a cell membrane. Forexample, Robert et al., Protein Eng. Des. Sel. (2008) Oct 16;S1741-0134, published online ahead, describe the use of chimericrecombinant Fab (rFab) and single chain fragments (scFvs) of monoclonalantibody WO-2 which recognizes an epitope in the N-terminal region ofAβ. The engineered fragments were able to (i) prevent amyloidfibrillization, (ii) disaggregate preformed Aβ1-42 fibrils and (iii)inhibit Aβ1-42 oligomer-mediated neurotoxicity in vitro as efficientlyas the whole IgG molecule. The perceived advantages of using small Faband scFv engineered antibody formats which lack the effector functioninclude more efficient passage across the blood-brain barrier andminimizing the risk of triggering inflammatory side reactions.Furthermore, besides scFv and single-domain antibodies retain thebinding specificity of full-length antibodies, they can be expressed assingle genes and intracellularly in mammalian cells as intrabodies, withthe potential for alteration of the folding, interactions,modifications, or subcellular localization of their targets; see forreview, e.g., Miller and Messer, Molecular Therapy 12 (2005), 394-401.

In a different approach Muller et al., Expert Opin. Biol. Ther. (2005),237-241, describe a technology platform, so-called ‘SuperAntibodyTechnology’, which is said to enable antibodies to be shuttled intoliving cells without harming them. Such cell-penetrating antibodies opennew diagnostic and therapeutic windows. The term ‘TransMabs’ has beencoined for these antibodies.

In a further embodiment, co-administration or sequential administrationof other neuroprotective agents useful for treating a synucleinopathicdisease may be desirable. In one embodiment, the additional agent iscomprised in the pharmaceutical composition of the present invention.Examples of neuroprotective agents which can be used to treat a subjectinclude, but are not limited to, an acetylcholinesterase inhibitor, aglutamatergic receptor antagonist, kinase inhibitors, HDAC inhibitors,anti-inflammatory agents, divalproex sodium, or any combination thereof.Examples of other neuroprotective agents that may be used concomitantwith pharmaceutical composition of the present invention are describedin the art; see, e.g. international application WO2007/011907. In oneembodiment, the additional agent is dopamine or a dopamine receptoragonist.

In a further embodiment of the present invention the α-synuclein bindingmolecules, in particular antibodies of the present invention may also beco-administered or administered before or after transplantation therapywith neural transplants or stem cell therapy useful for treating asynucleinopathic disease. Such approaches with transplants of embryonicmesencephalic neurons have been performed in patients with Parkinson'sdisease with the aim of replacing the neurons that are lost in thedisease and reinstating dopaminergic neurotransmission in the striatum.After 11-16 years post transplantation, the grafted neurons were foundto contain Lewy bodies and Lewy neurites. This spread of α-synucleinpathology from the host to the grated tissues may be prevented byco-administration of α-synuclein binding molecules, in particularantibodies of the present invention.

A therapeutically effective dose or amount refers to that amount of theactive ingredient sufficient to ameliorate the symptoms or condition.Therapeutic efficacy and toxicity of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED₅₀ (the dose therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose lethal to 50% of the population). Thedose ratio between therapeutic and toxic effects is the therapeuticindex, and it can be expressed as the ratio, LD₅₀/ED₅₀. Preferably, thetherapeutic agent in the composition is present in an amount sufficientto restore or preserve normal behavior and/or cognitive properties incase of PD, DLB or other synucleinopathic diseases.

From the foregoing, it is evident that the present invention encompassesany use of an α-synuclein binding molecule comprising at least one CDRof the above described antibody, in particular for diagnosing and/ortreatment of a synucleinopathic disease as mentioned above, particularlyParkinson's disease. Preferably, said binding molecule is an antibody ofthe present invention or an immunoglobulin chain thereof. In addition,the present invention relates to anti-idiotypic antibodies of any one ofthe mentioned antibodies described hereinbefore. These are antibodies orother binding molecules which bind to the unique antigenic peptidesequence located on an antibody's variable region near theantigen-binding site and are useful, e.g., for the detection ofanti-α-synuclein antibodies in sample of a subject.

In another embodiment the present invention relates to a diagnosticcomposition comprising any one of the above described α-synucleinbinding molecules, antibodies, antigen-binding fragments,polynucleotides, vectors or cells of the invention and optionallysuitable means for detection such as reagents conventionally used inimmuno or nucleic acid based diagnostic methods. The antibodies of theinvention are, for example, suited for use in immunoassays in which theycan be utilized in liquid phase or bound to a solid phase carrier.Examples of immunoassays which can utilize the antibody of the inventionare competitive and non-competitive immunoassays in either a direct orindirect format. Examples of such immunoassays are the radioimmunoassay(RIA), the sandwich (immunometric assay), flow cytometry and the Westernblot assay. The antigens and antibodies of the invention can be bound tomany different carriers and used to isolate cells specifically boundthereto. Examples of well known carriers include glass, polystyrene,polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran,nylon, amyloses, natural and modified celluloses, polyacrylamides,agaroses, and magnetite. The nature of the carrier can be either solubleor insoluble for the purposes of the invention. There are many differentlabels and methods of labeling known to those of ordinary skill in theart. Examples of the types of labels which can be used in the presentinvention include enzymes, radioisotopes, colloidal metals, fluorescentcompounds, chemiluminescent compounds, and bioluminescent compounds; seealso the embodiments discussed hereinabove.

By a further embodiment, the α-synuclein binding molecules, inparticular antibodies of the present invention may also be used in amethod for the diagnosis of a disorder in an individual by obtaining abody fluid sample from the tested individual which may be a bloodsample, a lymph sample or any other body fluid sample and contacting thebody fluid sample with an antibody of the instant invention underconditions enabling the formation of antibody-antigen complexes. Thelevel of such complexes is then determined by methods known in the art,a level significantly higher than that formed in a control sampleindicating the disease in the tested individual. In the same manner, thespecific antigen bound by the antibodies of the invention may also beused. Thus, the present invention relates to an in vitro immunoassaycomprising the binding molecule, e.g., antibody or antigen-bindingfragment thereof of the invention.

In this context, the present invention also relates to meansspecifically designed for this purpose. For example, a antibody-basedarray may be used, which is for example loaded with antibodies orequivalent antigen-binding molecules of the present invention whichspecifically recognize α-synuclein. Design of microarray immunoassays issummarized in Kusnezow et al., Mol. Cell Proteomics 5 (2006), 1681-1696.Accordingly, the present invention also relates to microarrays loadedwith α-synuclein binding molecules identified in accordance with thepresent invention.

In one embodiment, the present invention relates to a method ofdiagnosing a synucleinopathic disease in a subject, the methodcomprising:

-   -   (a) assessing a level of α-synuclein in a sample from the        subject to be diagnosed with an antibody of the present        invention, an α-synuclein binding fragment thereof or an        α-synuclein binding molecule having substantially the same        binding specificities of any one thereof; and    -   (b) comparing the level of the α-synuclein to a reference        standard that indicates the level of the α-synuclein in one or        more control subjects, wherein a difference or similarity        between the level of the α-synuclein and the reference standard        indicates that the subject has Parkinson's disease.

The subject to be diagnosed may be asymptomatic or preclinical for thedisease. Preferably, the control subject has a synucleinopathic disease,for example PD, DLB or LBVAD, wherein a similarity between the level ofα-synuclein and the reference standard indicates that the subject to bediagnosed has a synucleinopathic disease. Alternatively, or in additionas a second control the control subject does not have a synucleinopathicdisease, wherein a difference between the level of α-synuclein and thereference standard indicates that the subject to be diagnosed has asynucleinopathic disease. Preferably, the subject to be diagnosed andthe control subject(s) are age-matched. The sample to be analyzed may beany body fluid suspected to contain α-synuclein, for example a blood,CSF, or urine sample

The level of α-synuclein may be assessed by any suitable method known inthe art comprising, e.g., analyzing α-synuclein by one or moretechniques chosen from Western blot, immunoprecipitation, enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescentactivated cell sorting (FACS), two-dimensional gel electrophoresis, massspectroscopy (MS), matrix-assisted laser desorption/ionization-time offlight-MS (MALDI-TOF), surface-enhanced laser desorption ionization-timeof flight (SELDI-TOF), high performance liquid chromatography (HPLC),fast protein liquid chromatography (FPLC), multidimensional liquidchromatography (LC) followed by tandem mass spectrometry (MS/MS), andlaser densitometry. Preferably, said in vivo imaging of α-synucleincomprises positron emission tomography (PET), single photon emissiontomography (SPECT), near infrared (NIR) optical imaging or magneticresonance imaging (MRI).

Methods of diagnosing a synucleinopathic disease such as Parkinson'sdisease or Lewy body disease, monitoring a synucleinopathic diseaseprogression, and monitoring a synucleinopathic disease treatment usingantibodies and related means which may be adapted in accordance with thepresent invention are also described in international applicationWO2007/011907. Similarly, antibody based detection methods forα-synuclein are described in international applications WO99/50300,WO2005/047860, WO2007/021255 and WO2008/103472, the disclosure contentof all being incorporated herein by reference. Those methods may beapplied as described but with an α-synuclein specific antibody, bindingfragment, derivative or variant of the present invention.

These and other embodiments are disclosed and encompassed by thedescription and examples of the present invention. Further literatureconcerning any one of the materials, methods, uses and compounds to beemployed in accordance with the present invention may be retrieved frompublic libraries and databases, using for example electronic devices.For example the public database “Medline” may be utilized, which ishosted by the National Center for Biotechnology Information and/or theNational Library of Medicine at the National Institutes of Health.Further databases and web addresses, such as those of the EuropeanBioinformatics Institute (EBI), which is part of the European MolecularBiology Laboratory (EMBL) are known to the person skilled in the art andcan also be obtained using internet search engines. An overview ofpatent information in biotechnology and a survey of relevant sources ofpatent information useful for retrospective searching and for currentawareness is given in Berks, TIBTECH 12 (1994), 352-364.

The above disclosure generally describes the present invention. Unlessotherwise stated, a term as used herein is given the definition asprovided in the Oxford Dictionary of Biochemistry and Molecular Biology,Oxford University Press, 1997, revised 2000 and reprinted 2003, ISBN 019 850673 2. Several documents are cited throughout the text of thisspecification. Full bibliographic citations may be found at the end ofthe specification immediately preceding the claims. The contents of allcited references (including literature references, issued patents,published patent applications as cited throughout this application andmanufacturer's specifications, instructions, etc) are hereby expresslyincorporated by reference; however, there is no admission that anydocument cited is indeed prior art as to the present invention.

A more complete understanding can be obtained by reference to thefollowing specific examples which are provided herein for purposes ofillustration only and are not intended to limit the scope of theinvention.

EXAMPLES

The examples which follow further illustrate the invention, but shouldnot be construed to limit the scope of the invention in any way. Thefollowing experiments in Examples 1 and 2 are illustrated and describedwith respect to antibody NI-202.3G12, NI-202.12F4, and NI-202.3D8 ascloned, i.e. containing primer induced mutations at the very N-terminiof the framework 1 Ig-variable regions and not being adjusted to thegerm line (GL) sequences of human variable heavy and light chains; seeFIG. 1. However, the other antibodies of the NI-202 series, inparticular those with the adjusted GL sequences are structurally similarand thus may be expected to provide comparable results. These antibodieswere expressed as human IgG1 molecules. The experiments in examples 3and 4 are illustrated and described with respect to antibody NI-202.12F4with primer induced mutations at the N-termini of the Ig-variableregions being adjusted to the germ line (GL) sequences of human variableheavy and light chains; see FIG. 1. This antibody was expressed as achimeric molecule where the adjusted human variable domains were fusedto mouse IgG2a constant regions to allow for chronic dosing studies intransgenic mouse models without to induce a mouse anti-human immuneresponse.

Material and Methods

Detailed descriptions of conventional methods, such as those employedherein can be found in the cited literature. Unless indicated otherwisebelow, identification of α-synuclein-specific B cells and molecularcloning of α-synuclein antibodies displaying specificity of interest aswell as their recombinant expression and functional characterization hasbeen or can be performed as described in the Examples and SupplementaryMethods section of international application PCT/EP2008/000053 publishedas WO2008/081008, the disclosure content of which is incorporated hereinby reference in its entirety.

Purification of Antigen

Recombinant His-α-synuclein was obtained by recombinant expression inEscherichia coli and subsequent purification using heat inducedprecipitation, Nickel affinity-, anion exchange- and sizeexclusion-chromatography. For example, a DNA construct comprising thecDNA encoding α-synuclein under the control of the T7 promotor was usedto transform an appropriate Escherichia coli strain such as BL21(DE3)and expression of 200 ml cell culture was induced by the addition of 1mM isopropyl β-D-thiogalactopyranoside (IPTG). Cells were harvestedafter 4 hrs induction at 37° C. and then resuspended in 20 ml 50 mMTris, 150 mM NaCl pH 8, followed by sonification. After boiling for 15min, the heat resistant 17000 g supernatant was collected. Similar,heat-resistant 17000 g supernatant from mock Escherichia coli wascollected. After heat resistant 17000 g supernatant (20 ml) fromEscherichia coli expressing His-tagged α-synuclein was adjusted to 50 mMTris, 300 mM NaCl, 20 mM Imidazole, pH 8, it was loaded onto a HisTrapHP lml (GE Life Science) column and HIS-α-synuclein was eluted with an30-500mM imidazole gradient. Fractions containing HIS-α-synuclein werepooled and then diluted 1:10 with 50 mM Tris pH 8. Diluted pooledfractions were applied to a HiTrap Q HP lml (GE Life Science) column andbound proteins were eluted in a 30-1000 mM NaCl gradient. Finally,eluates containing HIS-α-synuclein were further purified using highperformance gel filtration (Superdex 200 10/300 GL). This purificationprocedure yields HIS-α-synuclein with a purity grade of around 99% asestimated by SDS-PAGE and Coomassie staining. Concentration of purifiedprotein has been determined using a BCA assay (Pierce).

α-Synuclein Antibody Screening

96 well half area Microplates (Corning) were coated with purifiedHIS-α-synuclein or α-synuclein (rPeptide) at a standard concentration of2 μg/ml in coating buffer (PBS pH 9.6) overnight at 4° C. Plates werewashed in PBS-T pH 7.6 and non-specific binding sites were blocked for 1hr at RT with PBS-T containing 2% BSA (Sigma, Buchs, Switzerland). Bcell conditioned medium was preabsorbed for 1 hr at RT with 10%Heat-resistant E. coli proteins in 1% BSA. This preabsorption step hadbeen developed after several previous attempts of ELISA screening wereunsuccessful in identifying human α-synuclein specific antibodies. Thus,fortunately it turned out that preabsorption of the ELISA plate withheat-resistant E. coli proteins excludes screening for false positivehits such as sticky antibodies and antibodies directed against Ecoliprotein contaminations probably present in purified recombinantα-synuclein samples. Preabsorbed medium was then transferred from memoryB cell culture plates to ELISA plates and incubated for 2 hrs at RT.ELISA plates were washed in PBS-T and then incubated with horse radishperoxidase (HRP)-conjugated donkey anti-human IgG (Fcγ fragmentspecific) polyclonal antibodies. After washing with PBS-T, binding ofhuman antibodies was determined by measurement of HRP activity in astandard colorimetric assay.

Molecular Cloning of α-Synuclein Antibodies

Samples containing memory B cells were obtained from volunteers >60years of age. All volunteers had in common to lack any sign ofParkinsonism. Living B cells of selected memory B cell cultures areharvested and mRNA is prepared. Immunoglobulin heavy and light chainsequences are then obtained using Ig-framework 1 specific primers forall human variable heavy and light chain families as 5′ primers incombination with primers specific for all human J segments (heavy andkappa light chain) and C segments (lambda light chain) as 3′primers(Marks et al., Mol. Biol. 222 (1991), 581-597; de Haard et al., J. Biol.Chem. 26 (1999), 18218-18230).

Identification of the antibody clone with the desired specificity isperformed by re-screening on ELISA upon recombinant expression ofcomplete antibodies. Recombinant expression of complete human IgG1antibodies or chimeric IgG2a antibodies is achieved upon insertion ofthe variable heavy and light chain sequences “in the correct readingframe” into expression vectors that complement the variable regionsequence with a sequence encoding a leader peptide at the 5′-end and atthe 3′-end with a sequence encoding the appropriate constant domain(s).To that end the primers contained restriction sites designed tofacilitate cloning of the variable heavy and light chain sequences intoantibody expression vectors. Heavy chain immunoglobulin are expressed byinserting the immunoglobulin heavy chain RT-PCR product in frame into aheavy chain expression vector bearing a signal peptide and the constantdomains of human immunoglobulin gamma 1 or mouse immunoglobulin gamma2a. Kappa light chain immunoglobulin is expressed by inserting the kappalight chain RT-PCR-product of NI-202.3D8 in frame into a light chainexpression vector providing a signal peptide and the constant domain ofhuman kappa light chain immunoglobulin. NI-202.12F4 and NI-202.3G12lambda light chain immunoglobulins are expressed by inserting the lambdalight chain RT-PCR-product in frame into a lambda light chain expressionvector providing a signal peptide and the constant domain of human ormouse lambda light chain immunoglobulin.

Functional recombinant monoclonal antibodies are obtained uponco-transfection into HEK293 or CHO cells (or any other appropriaterecipient cell line of human or mouse origin) of an Ig-heavy-chainexpression vector and a kappa or lambda Ig-light-chain expressionvector. Recombinant human monoclonal antibody is subsequently purifiedfrom the conditioned medium using a standard Protein A columnpurification. Recombinant human monoclonal antibody can produced inunlimited quantities using either transiently or stably transfectedcells. Cell lined producing recombinant human monoclonal antibody can beestablished either by using the Ig-expression vectors directly or byre-cloning of Ig-variable regions into different expression vectors.Derivatives such as F(ab), F(ab)₂ and scFv can also be generated fromthese Ig-variable regions.

Antibodies

Rabbit polyclonal pan synuclein antibody (Abcam), mouse monoclonal LB509α-synuclein specific antibody (Invitrogen), antibody Syn211 (Sigma) andClone 42 (BD Biosciences) were used according to manufacturer'sprotocol. Recombinant human α-synuclein antibodies NI202.3G12,NI202.12F4 and NI-202.3D8 are antibodies of this invention. They wereexpressed in HEK293 or CHO cells and then conditioned media was directlyused in subsequent applications unless otherwise stated. In Examples 3to 5 purified recombinant antibodies of the present invention were used.

Direct ELISA

cAntigens were coated at indicated concentration in PBS pH 9.6 onto 96well half area microplates (Corning) overnight at 4° C. Plates werewashed in PBS-T pH 7.6 and non-specific binding sites were blocked for 1hr at RT with PBS-T containing 2% BSA (Sigma). Probes (Primaryantibodies) were then transferred to wells and incubated for 2 hrs atRT. After washing in PBS-T pH 7.6, wells were incubated with horseradish peroxidase (HRP)-conjugated polyclonal anti-human (forrecombinant human antibodies), anti-rabbit (for pan synuclein antibody)or anti-mouse (for LB509 or Syn211) secondary antibodies for 1 hr at RT.After rigorous washing in PBS-T, binding of probes was determined bymeasurement of HRP activity in a standard colorimetric assay using3,3′,5,5′-tetramethylbiphenyl-4,4′-diamine (Sigma) as chromogenicsubstrate.

Western Blotting and Coomassie Protein Staining

To assess binding to human and mouse α-synuclein, frozen brains of wildtype or α-synuclein transgenic mice were homogenized in PBS (10 ml/g wetweight) using a Dounce homogenizer. The extract was spun at 100,000 gand the supernatant was designated soluble fraction. Soluble fraction orrecombinant proteins (750 ng) were mixed with loading dye, heated at 65°C. for 10 min and 0.75 pg was loaded per lane and separated on a 4-20%Tris-Glycine SDS-PAGE. Gels were either stained in 0.025% CoomassieBrilliant blue R 250 (Fluka) solution or electroblotted tonitrocellulose transfer membrane. Blots were then incubated with primaryantibody for 2 hrs. Binding of primary antibodies was revealed usingsecondary anti human antibodies conjugated with HRP. Blots weredeveloped using ECL plus Western Blotting Detection Reagents (GEHealthcare).

To assess binding to α-synuclein monomers and aggregates, brain extractswere prepared in PBS containing 0.5% Triton X100 followed bycentrifugation at 1000 g for 5 min. Supernatants were separated by 4-12%Bis-Tris NuPAGE gel electrophoresis and analyzed by WB.

Pole Test

Mice are tested at the beginning of the dark phase when they are mostactive. The pole is made of a wooden stick with 50 cm length and 1 cmwidth covered with cloth to facilitate climbing. The base of the pole isplaced in the home cage of the mouse. The mouse is placed on the top ofthe pole and the time to orient downwards and time to climb down to thehome cage is recorded over 5 trials with 30 min intertrial intervals.The best performance trial is analyzed.

Elevated Plus Maze Test

Mice are tested at the beginning of the dark phase when they are mostactive. Testing is performed in dim light (40 lux). The elevated plusmaze consists of two open and two closed arms (arm length: 30 cm; width:5 cm). Open arms have a small 1 cm edge and the closed arms are borderedby a 15 cm wall. At the beginning of the task, mice are placed in thecenter of the elevated plus maze facing an open arm. Mice arevideo-tracked while exploring the maze for 5 min. The time spent in theopen and closed arms and the distance covered are measured and analyzed.

Transgenic Mice

B6;C3-Tg(Prnp-SNCA*A53T)83Vle/J (Giasson et al., Neuron. 34 (2002),521-533) transgenic α-synuclein mice and corresponding wild type micewere kept under standard housing conditions on a reversed 12 h:12 hlight/dark cycle with free access to food and water. The treatmentgroups were balanced for age and gender.

Determination of NI-202.12F4 and α-Synuclein Levels in Mouse Plasma

NI-202.12F4 plasma levels were determined using a direct α-synucleinELISA using recombinant NI-202.12F4 of known concentration as standard.For determination of human α-synuclein levels in plasma a sandwich ELISAwas applied (Invitrogen, USA).

Example 1 Identification of Human α-Synuclein-Specific Antibodies withDifferent Epitope Specificities

α-synuclein is a 140 amino acids (aa) long natively unfolded proteinthat is composed of three domains. These are the N-terminal amphipathicrepeat region (1-60 aa), the center region (61-95 95aa) and the acidicC-terminal region (96-140). To further understand the specificity ofrecombinant human α-synuclein antibodies, the domain of α-synuclein thatcontains the recognition sequence was determined. α-synucleintruncations 1-60 aa, 1-95 aa, 61-140 aa and 96-140 aa were coated atequal concentration onto ELISA plates and recombinant human α-synucleinautoantibodies NI-202.3G12 and NI-202.12F4 were then probed for bindingto these truncations.

Interestingly, NI-202.3G12 does only bind to coated full lengthα-synuclein but not to any of the four tested truncations (FIG. 2A).This suggests that the recognition epitope of NI-202.3G12 is astructural motif rather than a linear primary recognition sequence.

On the other hand NI-202.12F4 binds to α-synuclein fragments comprisingaa 1-60 (FIG. 2B) but not to N-terminally truncated fragments comprisingamino acids 61-140 or 96-140. This shows that its epitope is localizedwithin the N-terminus. Notably, characterization of mouse monoclonalantibodies which selectively bind α-synuclein in pathologicalinclusions, revealed that their epitopes are within the very N-terminalsegment (Waxman et al., Acta Neuropathol. 116 (2008), 37-46.

In addition, preliminary results of direct ELISA assays performed withantibody NI-202.3D8 revealed that NI-202.3D8 specifically recognizes theC-terminus of α-synuclein, in particular amino acids 96-140. Deletion ofkey amino acids 125-140 within the C-terminal domain greatly altersα-synuclein aggregation and in the brains of patients with Dementia withLewy body disease (LBD) as well as in transgenic animal models, there isabundant accumulation of C-terminal α-synuclein fragments. These studiessuggest that antibodies capable of recognizing the C-terminal regionmight have potential therapeutic effects; see Masliah et al., Neuron, 46(2005), 857-868.

To exclude that C-terminal α-synuclein comprising truncations are notefficiently coated onto ELISA plates, epitope mapping of the mousemonoclonal alpha synuclein antibody LB509 (Baba et al., Am. J. Pathol.152 (1998), 879-884) was performed. LB509 binds to a C-terminal epitope(Jakes et al., Neuroscience Letters 269 (1999), 13-16). As shown in FIG.2C, this study confirms the C-terminal epitope of LB509 and thusconfirms efficient coating of C-terminal α-synuclein fragments. Inconclusion, epitope mapping of recombinant human α-synuclein antibodiesin the experiments performed in accordance with the present inventionshows that these antibodies are directed against different epitopesincluding conformational epitopes and potential pathological structuresin the N-terminus and the C-terminus.

Example 2 The Human Antibodies are Specific for α-Synuclein

α-, β- and γ-synuclein are highly homologues proteins that arepredominantly expressed in the nervous system, skeletal muscle andheart. However, only abnormal α-synuclein is linked to a broad spectrumof CNS diseases while β-synuclein is suggested to be an inhibitor ofα-synuclein aggregation and may protect the central nervous system fromthe neurotoxic effects of α-synuclein. Thus (therapeutic) antibodiesagainst pathological α-synuclein variants preferentially do not crossreact with β- and γ-synuclein. In order to support specificity andpotential therapeutic use of recombinant human anti-α-synucleinantibodies, the candidate antibodies were probed for α-, β- andγ-synuclein binding in a direct ELISA assay and by Western blotting(WB). First, recombinant α-, β- and γ-synuclein have been coated ontoELISA plates at equal coating concentration (2 μg/ml) and were theneither incubated with a pan synuclein control antibody or recombinanthuman α-synuclein antibodies. The pan synuclein antibody reacts with allthree synuclein proteins coated on ELISA plates (FIG. 3A). Thenrecombinant human α-synuclein antibodies were probed for specificα-synuclein binding. Both NI-202.3G12 and NI-202.12F4 react with coatedα-synuclein but not β- and γ-synuclein (FIGS. 3A and 3B).

Similar, the specificity of the recombinant antibodies was alsoinvestigated using WB analysis. Coomassie protein staining confirms thatequal concentrations of all three synuclein proteins have been appliedto SDS PAGE analysis (FIG. 4A). WB analysis then shows that NI-202.12F4selectively binds to α-synuclein (FIG. 4B) whereas without primaryantibody no signal was detected (FIG. 4C). NI-202.3G12 binding toα-synuclein on WB was undetectable. This is in agreement with astructural epitope rather than a linear recognition sequence ofNI-202.3G12. These findings demonstrate that recombinant human alphasynuclein antibodies described in the present invention can be highlyspecific.

All subsequent experiments were performed with the NI-202.12F4 mousechimeric antibody.

Example 3 Binding Specificities of Antibody NI-202.12F4

Specific Binding to Human α-Synuclein with a Preference for Aggregatedα-Synuclein Species

To assess the binding of NI-202.12F4 to human and mouse α-synuclein,brain extracts were prepared from wild type and α-synuclein A53Ttransgenic mice and antibody binding was analyzed by Western blotting.NI-202.12F4 detects a prominent band corresponding to α-synuclein inbrain extracts from human A53T α-synuclein transgenic mice while suchband is virtually absent in brain extracts from wild type mice (FIG.5A), suggesting specific binding to human but not mouse α-synuclein.Similar results were obtained with the commercially available LB509antibody (Jakes et al., Neuroscience Letters 269 (1999), 13-16) directedagainst an epitope specific for human α-synuclein. In contrast, thecommercially available antibody clone 42 (van der Putten et al., J.Neurosci. 20 (2000), 6021-6029) which was reported to bind α-synucleinof both species, detected human as well as mouse α-synuclein protein.These data suggest that NI-202.12F4 preferentially binds to humanα-synuclein.

To assess the binding of NI-202.12F4 to physiological forms as well aspathological aggregates of human α-synuclein, cortical brain extractsfrom a healthy control subject and a dementia with Lewy bodies (DLB)patient as well as extracts from wild type mice and human A30Pα-synuclein transgenic mice brain (Kahle et al. J. Neurosci. 20 (2000),6365-73) were prepared, separated by SDS gel electrophoresis andanalyzed by Western blotting. NI-202.12F4 detects with high sensitivitya prominent smear reflecting oligomeric and fibrillar forms ofα-synuclein aggregates in DLB and A30P α-synuclein transgenic brainextract but not in healthy control and wild type control extracts (FIG.5B). In contrast minimal binding was observed to monomeric forms ofα-synuclein in human or wild type mouse tissues and moderate binding inA30P α-synuclein transgenic brain extracts highly overexpressing theα-synuclein protein. In contrast, clone 42 antibody showed the oppositebinding pattern in the Western blot analysis, detecting monomeric formsand α-synuclein fragments with a high sensitivity and poorly binding toaggregated α-synuclein species. These results suggest that NI-202.12F4preferentially binds to pathological aggregates of α-synuclein such asoligomers and fibrils over physiological α-synuclein monomers.

NI-202.12F4 Binds to Wild Type and Disease Causing Mutants of HumanAlpha Synuclein with High Affinity

The half maximal effective concentration (EC50) was determined for wildtype as well as disease causing human α-synuclein mutants using a directα-synuclein ELISA. High affinity binding was observed for the wild typeas well as the A30P, E46K and A53T mutant forms of human α-synuclein(FIG. 6).

Recombinant NI-202.12F4 Preferentially Binds to Pathological Forms ofα-Synuclein in Brain.

Binding of NI-202.12F4 to α-synuclein was further characterized byimmunohistochemical staining of brain sections from α-synucleintransgenic mice and patients with neuropathologically confirmedParkinson's Disease or dementia with Lewy bodies. NI-202.12F4 showsprominent staining of α-synuclein pathology including Lewy body and Lewyneurite like inclusion as well as small somatodendritic and synapticaccumulations of α-synuclein in free-floating sections from transgenicmice expressing either human A53T (FIG. 7A) or A30P α-synuclein (FIG.7B) as well as in human brain tissues of Parkinson's disease anddementia with Lewy bodies (FIG. 7C, D). In contrast to the commerciallyavailable Syn211 antibody (Giasson et al., J. Neurosci. Res. 59 (2000),528-33) which detects also physiological synaptic α-synuclein with ahigh sensitivity (FIG. 7E), NI-202.12F4 binds preferentially topathological aggregates of α-synuclein as exemplified byimmunohistochemical staining of human A30P α-synuclein transgenic mice(FIG. 7B). Binding of NI-202.12F4 is virtually absent on brain sectionsof wild type mice (FIG. 7F) comparable to secondary antibody onlycontrol staining (FIG. 7G) confirming the preferential binding to humanversus mouse α-synuclein as was observed in the Western blot analysis(FIG. 5). In contrast, the commercially available clone 42 antibody (vander Putten et al., J. Neurosci. 20 (2000), 6021-6029) which was reportedto bind α-synuclein of human as well as mouse origin, showed prominentsynaptic staining of mouse α-synuclein protein in brain sections of wildtype mice (FIG. 7H).

NI-202.12F4 shows preferential binding to human α-synuclein at highcoating concentrations pointing to a conformational epitope

The half maximal effective concentration (EC50) indicating the potencyof an antibody was determined for low and high coating concentrations ofrecombinant α-synuclein using a direct α-synuclein ELISA. High affinitybinding of recombinant NI-202.12F4 with an EC50 of ˜100 pM was observedfor high coating densities of α-synuclein protein (20 μg/ml). At lowercoating concentrations of α-synuclein, a sharp drop in affinity wasobserved, with a corresponding increase in EC50 by close to 100-fold(FIG. 8A). In contrast, the commercially available Syn211 antibody(Giasson et al., J. Neurosci. Res. 59 (2000), 528-33) directed against alinear epitope within the α-synuclein C-terminus showed no decrease inbinding affinity at lower coating densities of alpha synuclein (FIG.8B). These findings suggest that NI-202.12F4 preferentially targets astructural epitope of α-synuclein that is formed at high concentrationsof α-synuclein. The results are consistent with the immunohistochemicalbinding characteristics of NI-202.12F4 that suggest a preference forpathologic conformations of α-synuclein aggregates such as α-synucleinfibrils or oligomers. As was observed for full-length α-synuclein,NI-202.12F4 binding to truncated α-synuclein 1-60 shows equivalentdependence on the coating concentration pointing to a conformationalepitope that is contained within amino acids 1-60 of the α-synucleinprotein (FIG. 8C). It can however not be excluded that amino acids60-140 of α-synuclein can influence the formation of the N-terminalepitope. Notably, the N-terminal amino acids 1-60 of human α-synucleincontaining the A53T mutation are 100% identical to the N-terminus ofmouse α-synuclein. Thus the observed preference for human α-synucleinversus the mouse ortholog could be due to an influence of the C-terminalpart of synuclein on the accessibility or formation of the preferredNI-202.12F4 epitope at the N-terminus. Epitope mapping studies usingsmaller N-terminal derived fragments of α-synuclein (amino acids 1-20;21-40; 41-60; 11-30; 31-50) revealed no binding of the NI-202.12F4antibody to any of the peptides, suggesting that these regions are notsufficient for binding of NI-202.12F4 (FIG. 8D).

Example 4: Recombinant Human-Derived Antibody Against Alpha-SynucleinImproves Motor Performance and Elevated plus Maze Behavior in aTransgenic Mouse Model of Parkinson's Disease

To assess the pharmacological effects of NI-202.12F4 treatment, 10.5months old transgenic mice overexpressing the human A53T α-synucleintransgene (Giasson et al., Neuron 34 (2002), 521-533) were treatedweekly i.p. with 5 mg/kg of recombinant chimeric NI-202.12F4 antibody orvehicle control for a total of 4 months. After 2 months of treatment,motor performance was evaluated in the pole test. NI-202.12F4 treatedmice showed a significant improvement in motor performance compared tothe vehicle treated group with significantly reduced time to turn on thepole (Tturn; p<0.001; n=17-19 animals per group) as well as total timeto descend into the home cage (Ttotal; p<0.05; n=17-19 animals pergroup) as is shown in FIG. 9. In a second behavior test the treatmenteffects on elevated plus maze performance was analyzed. α-synuclein A53Ttransgenic mice were previously reported to exhibit impaired elevatedplus maze behavior, spending more time in open arms compared to wildtype controls (George et al., Exp. Neurol., 210 (2008), 788-92). Asshown in FIG. 10 chronic treatment with NI-202.12F4 lead to asignificant improvement in elevated plus maze behavior in α-synucleinA53T transgenic animals. Antibody treated mice spend significantly lesstime and covered a significantly lower distance in open arms compared tovehicle treated animals (p<0.05; n=17-19 animals per group) while theactivity levels were equivalent for both groups. These resultsdemonstrate that chronic treatment with the NI-202.12F4 antibody leadsto significant improvements in motor performance and rescues abnormalelevated plus maze behavior in α-synuclein transgenic mouse models ofParkinson's disease.

Example 5 NI-202.12F4 Antibody Increases Plasma Synuclein Levels inSynuclein Transgenic Mouse Models of Parkinson's Disease

10.5 old month transgenic A53T α-synuclein transgenic mice were treatedweekly i.p. for 2 month with 5 mg/kg chimeric NI-202.12F4 or PBS. 24 hafter the last injection, plasma samples were prepared and the plasmaconcentrations of treatment antibody and human α-synuclein weredetermined by ELISA (FIG. 11). Plasma levels of human α-synuclein weresignificantly increased by more than 10 fold compared to vehicle treatedanimals (25±4.1 ng/ml for NI-202.12F4 treatment group vs. 1.9±1.2 ng/mlfor PBS group, p=0.0002) demonstrating pharmacodynamic modulation ofα-synuclein upon treatment with NI-202.12F4. Similar effects wereobserved upon acute antibody treatment in the A30P α-synucleintransgenic mice. As in the A53T and A30P α-synuclein transgenic modelshuman α-synuclein is predominantly expressed in the brain, the increasein circulating human α-synuclein could be due to a NI-202.12F4 mediatednet efflux of α-synuclein from brain to the periphery.

1-30. (canceled)
 31. A method of treating a synucleinopathic disease ina human subject in need thereof, the method comprising administering tothe human subject a therapeutically effective amount of an antibody thatbinds to human alphα-synuclein and comprises: (i) an immunoglobulinheavy chain comprising a heavy chain variable region (VH) comprising VHcomplementarity determining regions (CDRs) 1, 2, and 3 with the aminoacid sequences set forth in residues 31-35 of SEQ ID NO:9, residues50-68 of SEQ ID NO:9, and residues 101-102 of SEQ ID NO:9, respectively;and (ii) an immunoglobulin light chain comprising a light chain variableregion (VL) comprising VL CDRs 1, 2, and 3 with the amino acid sequencesset forth in residues 23-33 of SEQ ID NO:12, residues 49-55 of SEQ IDNO:12, and residues 88-98 of SEQ ID NO:12, respectively.
 32. The methodof claim 31, wherein (i) the VH consists of the amino acid sequence setforth in SEQ ID NO:9; and (ii) the VL consists of the amino acidsequence set forth in SEQ ID NO:12.
 33. The method of claim 31, wherein(i) the VL consists of the amino acid sequence set forth in SEQ IDNO:10; and (ii) the VL consists of the amino acid sequence set forth inSEQ ID NO:13.
 34. The method of claim 31, wherein the immunoglobulinheavy chain comprises a human IgG1 heavy chain constant region and theimmunoglobulin light chain comprises a human lambda light chain constantregion.
 35. The method of claim 33, wherein the immunoglobulin heavychain comprises a human IgG1 heavy chain constant region and theimmunoglobulin light chain comprises a human lambda light chain constantregion.
 36. The method of claim 31, wherein the synucleinopathic diseaseis Parkinson's disease, dementia with Lewy bodies, multiple systematrophy, Parkinson's disease dementia, or the Lewy body variant ofAlzheimer's disease.
 37. The method of claim 33, wherein thesynucleinopathic disease is Parkinson's disease, dementia with Lewybodies, multiple system atrophy, Parkinson's disease dementia, or theLewy body variant of Alzheimer's disease.
 38. The method of claim 31,wherein the synucleinopathic disease is Parkinson's disease.
 39. Themethod of claim 33, wherein the synucleinopathic disease is Parkinson'sdisease.
 40. The method of claim 34, wherein the synucleinopathicdisease is Parkinson's disease.
 41. The method of claim 35, wherein thesynucleinopathic disease is Parkinson's disease.
 42. An antibody orantigen-binding fragment thereof that binds to human alpha-synuclein andcomprises: an immunoglobulin heavy chain comprising a heavy chainvariable region (VH) comprising VH complementarity determining regions(CDRs) 1, 2, and 3 with the amino acid sequences set forth in residues31-35 of SEQ ID NO:9, residues 50-68 of SEQ ID NO:9, and residues101-102 of SEQ ID NO:9, respectively; and an immunoglobulin light chaincomprising a light chain variable region (VL) comprising VL CDRs 1, 2,and 3 with the amino acid sequences set forth in residues 23-33 of SEQID NO:12, residues 49-55 of SEQ ID NO:12, and residues 88-98 of SEQ IDNO:12, respectively, wherein the antibody or antigen-binding fragmentthereof comprises a detectable label selected from the group consistingof an enzyme, a radioisotope, a fluorescent compound, a chemiluminescentcompound, a bioluminescent compound, and a heavy metal.
 43. The antibodyor antigen-binding fragment thereof of claim 42, wherein (i) the VHconsists of the amino acid sequence set forth in SEQ ID NO:10; and (ii)the VL consists of the amino acid sequence set forth in SEQ ID NO:13.44. A pharmaceutical composition comprising the antibody orantigen-binding fragment thereof of claim 42, and a pharmaceuticallyacceptable carrier.
 45. A method for in vivo detection of α-synuclein ina human subject, the method comprising administering to the humansubject a composition comprising the antibody or antigen-bindingfragment thereof of claim 41, wherein α-synuclein is detected bypositron emission tomography (PET), single photon emission tomography(SPECT), near infrared (NIR) optical imaging, magnetic resonance imaging(MM), or a combination thereof.
 46. The method of claim 45, wherein thehuman subject has or is suspected of having Parkinson's disease,dementia with Lewy bodies, multiple system atrophy, Parkinson's diseasedementia, or the Lewy body variant of Alzheimer's disease.